Receptor-mediated uptake of an extracellular Bcl-xL fusion protein inhibits apoptosis

ABSTRACT

Apoptosis-modifying fusion polypeptides, and the corresponding nucleic acid molecules, are disclosed. Pharmaceutical compositions comprising these polypeptides, and the use of these polypeptides to modify apoptosis are also provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This is a divisional of U.S. application Ser. No. 09/639,245,filed Aug. 15, 2000, which claims the benefit of U.S. ProvisionalApplication No. 60/149,220, filed Aug. 16, 1999, both of which areincorporated herein by reference in their entirety.

FIELD

[0002] This invention relates to modification of the apoptotic responseof target cells, for instance target cells in a subject. Morespecifically, it relates to apoptosis-modifying fusion proteins with atleast two domains, one of which targets the fusion protein to a targetcell, and another of which modifies an apoptotic response of the targetcell.

BACKGROUND

[0003] Tissue and cell homeostasis in multicellular organisms is largelyinfluenced by apoptosis, the phenomenon of programmed cell death bywhich an intra- or extra-cellular trigger causes a cell to activate abiochemical “suicide” pathway. Morphological indicia of apoptosisinclude membrane blebbing, chromatin condensation and fragmentation, andformation of apoptotic bodies, all of which take place relatively earlyin the process of programmed cell death. Degradation of genomic DNAduring apoptosis results in formation of characteristic, nucleosomesized DNA fragments; this degradation produces a diagnostic ˜180 bpladdering pattern when analyzed by gel electrophoresis. A later step inthe apoptotic process is degradation of the plasma membrane, renderingapoptotic cells leaky to various dyes (e.g., trypan blue and propidiumiodide). Apoptotic cells are usually engulfed and destroyed early in thedeath process; thus, apoptosis tends not to be associated withinflammation caused by cytoplasm leakage, as is found in necrosis.

[0004] Various in vivo triggers can induce apoptosis; the paradigmatictrigger is a shortage of one or more necessary growth factors. Apoptosisplays a significant role in development of the neural system (reviewedin Cowan et al., Science 225:1258-1265, 1984; Davies, Development101:185-208, 1987; Oppenheim, Annu. Rev. Neurosci. 14:453-501, 1991) andlymphoid system (reviewed in Blackman et al., Science 248:1335-1341,1990; Rothenberg, Adv. Immunol. 51:85-214, 1992) of vertebrates. Systemdevelopment occurs through selective apoptotic extinction of certaincell populations.

[0005] In spite of much study, the molecular mechanisms of apoptosis arenot fully elucidated. It does appear, however, that different apoptosisinducers may trigger different apoptotic pathways. For instance, certainpathways are transcription-dependent, in that apoptosis requires thesynthesis of new proteins after stimulation by, for instance, withdrawalof growth factors. Staurosporine, a non-specific kinase inhibiter, incontrast, stimulates a transcription-independent pathway. Transcriptiondependent and independent pathways appear to share downstreamcomponents, including the ICE family of proteases (caspases). See Rubin,British Med. Bulle., 53:617-631, 1997, for a review of apoptosis inneurons; More general reviews include Ashkenazi and Dixit, Science281:1305-1308; Thomberry and Lazebnik, Science 281:1312-1316; and Adamsand Cory, Science 281:1322-1326.

[0006] Apoptosis is recognized as a gene-directed event, controlled by acomplex set of interacting gene products that inhibit or enhanceapoptosis (Williams and Smith, Cell 74:777-779, 1993; reviewed in White,Genes Dev. 10:1-15, 1996). Extensive effort is currently underway toidentify and characterize the genes involved in this process. The firstprotein characterized as influencing apoptosis was Bcl-2 (Cleary et al.,Cell 47:19-28, 1986; Tsujimoto and Croce, Proc. Natl. Acad. Sci. USA83:5214-5218, 1986). Since its discovery, several Bcl-2-related proteins(the Bcl-2 family of proteins) have been identified as being involved inregulation of apoptosis (White, Genes Dev. 10:1-15, 1996; Yang et al.,Cell 80:285-291, 1995). One such is Bcl-x, which is expressed in twodifferent forms, long (Bcl-x_(L)) and short (Bcl-x_(S)) (Boise et al.,Cell 74:597-608, 1993).

[0007] Bcl-x_(L) and certain other members of the Bcl-2 family are, likeBcl-2 itself, powerful inhibitors of cell death (the “anti-death” Bcl-2family members). Genetic overexpression of Bcl-2 has been shown to blockapoptosis in the nervous system of transgenic mice (Chen et al., Nature385:434-439, 1997; Henkart, Immunity 4:195-201, 1996;Lippincott-Schwartz et al., Cell 67:601-616, 1991; Hunziker et al., Cell67:617-627, 1991; Krajewski et al., Cancer Research 53:4701-4714, 1993;Martinou et al., Neuron 13:1017-1030, 1994).

[0008] Other members of the Bcl-2 protein family, including Bcl-x_(S),Bad and Bax, are potent enhancers of apoptosis and therefore toxic tocells (“pro-death” Bcl-2 family members). Though the mechanism ofapoptosis induction by these proteins remains unknown, it has beensuggested that Bad binding to Bcl-x_(L) may promote cell death (Yang etal., Cell 80:285-291, 1995; Zha et al., J Biol. Chem 272:24101-24104,1997) and that phosphorylation of Bad may prevent its binding toBcl-x_(L), thereby blocking cell death (Zha et al., J Biol. Chem.272:24101-24104, 1997; Zha et al., Cell 87:619-628, 1996).

[0009] In addition to its involvement in neuronal and lymphoid systemdevelopment and overall cell population homeostasis, apoptosis alsoplays a substantial role in cell death that occurs in conjunction withvarious disease and injury conditions. For instance, apoptosis isinvolved in the damage caused by neurodegenerative disorders, includingAlzheimer's disease (Barinaga, Science 281:1303-1304), Huntington'sdisease, and spinal-muscular atrophy. There is also a substantialapoptotic component to the neuronal damage caused during stroke episodes(reviewed in Rubin, British Med. Bulle., 53(3):617-631, 1997; andBarinaga, Science 281:1302-1303), and transient ischemic neuronalinjury, as in spinal cord injury. It would be of great benefit toprevent undesired apoptosis in various disease and injury situations.

[0010] Treatment with standard apoptosis inhibitory molecules, forinstance peptide-type caspase inhibitors (e.g., DEVD-type), thoughuseful for laboratory experiments where microinjection can be employed,has proven unsatisfactory for clinical work due to low membranepermeability of these inhibitors. Transfection of cells with variousnative proteins, including members of the Bcl-2 family of regulatoryproteins, has dual disadvantages. First, transfection is usually notcell-specific, and thus may disrupt apoptotic processes non-specificallyin all cells. Second, transfection tends to provide long termalterations in the apoptotic process, in that once a transgene isintegrated and functional in the genome of target cells, it may bedifficult to turn off. Especially in instances of stroke episodes ortransient ischemic neuronal injury, it would be more advantageous to beable to apply apoptosis regulation for short periods of time. Therefore,there is still a strong need to develop pharmaceutical agents thatovercome these disadvantages.

[0011] Cancer and other hyper-proliferative cell conditions can beviewed as inappropriate escape from appropriate cell death. As such, itwould be advantageous to be able to enhance apoptosis in certain ofthese cells to stop unregulated or undesired growth. Various attemptshave been made to selectively eliminate cancerous cells through the useof targeted immunotoxins (genetic or biochemical fusions between a toxicmolecule, for instance a bacterial toxin, and a targeting domainderived, typically from an antibody molecule).

[0012] One bacterial toxin that has been employed in attempts to killcancerous cells is diphtheria toxin (DT). Diphtheria toxin has threestructurally and functionally distinct domains: (1) a cell surfacereceptor binding domain (DTR), (2) a translocation domain (DTT) thatallows passage of the active domain across the cell membrane, and (3)the A (enzymatically active) chain that, upon delivery to a cell,ADP-ribosylates elongation factor 2 and thereby inactivates translation.Altering the receptor specificity of the diphtheria toxin has been usedto generate toxins that may selectively kill cancer cells in vitro(Thorpe et al., Nature 271:752-755, 1978) and in man (Laske et al.,Nature Medicine 3:1362-1368, 1997). Promising though they might haveseemed, these and similar hybrid immunotoxins have proven to besubstantially less effective at targeted cell death than the toxins fromwhich they were generated. This is perhaps due to difficulties intranslocation of the fusion protein into the target cell (Columbatti etal., J. Biol. Chem. 261:3030-3035, 1986). In addition, in vivo resultshave been particularly poor using such hybrid constructs (Fulton et al.,Fed. Proc. 461:1507, 1987).

[0013] It is to biological molecules that overcome deficiencies in theprior art that the present invention is directed.

SUMMARY OF THE DISCLOSURE

[0014] Disclosed herein are apoptosis-modifying fusion proteinsconstructed by fusing a protein, or an apoptosis-modifying fragment orvariant thereof, from the Bcl-2 protein family with a cell-binding,targeting domain such as one derived from a bacterial toxin. Using thisapproach, apoptosis-modifying fusion proteins can be deliveredeffectively throughout the body and targeted to select tissues andcells. In certain embodiments, fusing various cell-binding domains toBcl-2 family proteins (such as Bcl-x_(L) or Bad) allows targeting tospecific subsets of cells in vivo, permitting treatment and/orprevention of the cell-death related consequences of various diseasesand injuries. The delivery of other Bcl-2 homologues to the cell permitsregulation of cell viability either positively (using anti-death Bcl-2family members), or negatively (using pro-death members of the Bcl-2family).

[0015] The apoptosis-modifying fusion proteins disclosed herein havespecifiable cell-targeting and apoptosis-modifying activities. Thus,they may be used clinically to treat various disease and injuryconditions, through inhibition or enhancement of an apoptotic cellularresponse. For instance, apoptosis-inhibiting fusion proteins arebeneficial to minimize or prevent apoptotic damage that can be caused byneurodegenerative disorders (e.g., Alzheimer's disease, Huntington'sdisease, spinal-muscular atrophy), stroke episodes, and transientischemic neuronal injury (e.g., spinal cord injury). Theapoptosis-enhancing fusion proteins n can be used to inhibit cellgrowth, for instance uncontrolled cellular proliferation.

[0016] Accordingly, a first embodiment is a functionalapoptosis-modifying fusion protein capable of binding a target cell,having a first domain capable of modifying apoptosis in the target cell,and a second domain capable of specifically targeting the fusion proteinto the target cell. This fusion protein further integrates into orotherwise crosses a cellular membrane of the target cell upon binding tothat cell.

[0017] Certain embodiments will also include a linker between these twodomains. This linker will usually be at least 5 amino acids long, forexample between 5 and 100 amino acids in length, and may for instanceinclude the amino acid sequence shown in SEQ ID NO: 6. Appropriatelinkers may be 6, 7, or 8 amino acids in length, and so forth, includinglinkers of about 10, 20, 30, 40 or 50 amino acids long.

[0018] The apoptosis modifying fusion proteins may also include a thirddomain from one of the two original proteins, or from a third protein.This third domain may improve the fusion protein's ability to beintegrated into or otherwise cross a cellular membrane of the targetcell. An example of such a third domain is the translocation region(domain or sub-domain) of diphtheria toxin.

[0019] Target cells for the fusion proteins disclosed herein include,but are not limited to, neurons, lymphocytes, stem cells, epithelialcells, cancer cells, neoplasm cells, and others, including otherhyper-proliferative cells. The target cell chosen will depend on whatdisease or injury condition the fusion protein is intended to treat.

[0020] Receptor-binding domains may be derived from various cell-typespecific binding proteins, including for instance bacterial toxins(e.g., diphtheria toxin or anthrax toxin), growth factors (e.g.,epidermal growth factor), monoclonal antibodies, or single-chainantibodies derived from antibody genes. Further, variants or fragmentsof such proteins may also be used, where these fragments or variantsmaintain the ability to target the fusion protein to the appropriatetarget cell.

[0021] Further specific embodiments employ essentially the entireBcl-x_(L) protein as the apoptosis-modifying domain of the fusionprotein, or variants or fragments thereof that maintain the ability toinhibit apoptosis in a target cell to which the protein is exposed.Examples of such proteins are fusion proteins made of the Bcl-x_(L)protein, functionally linked to the diphtheria toxin receptor bindingdomain through a peptide linker of about six amino acids. One suchprotein is Bcl-x_(L)-DTR, which consists of Bcl-x_(L) and DTR, withoutthe translocation domain of diphtheria toxin. The nucleotide sequence ofthis fusion protein is shown in SEQ ID NO: 1, and the correspondingamino acid sequence in SEQ ID NOs: 1 and 2.

[0022] Another such example is LF_(n)-Bcl-x_(L), which includes theamino terminal portion (residues 1-255) of mature anthrax lethal factor(LF), coupled to residues 1-209 of Bcl-x_(L). The nucleotide sequence ofthis fusion protein is shown in SEQ ID NO: 7, and the correspondingamino acid sequence in SEQ ID NOs: 7 and 8.

[0023] Also encompassed are fusion proteins wherein theapoptosis-modifying domain is an apoptosis-enhancing domain. Suchdomains include the various pro-death members of the Bcl-2 family ofproteins, for instance Bad, and variants or fragments thereof thatenhance apoptosis in a target cell. A specific appropriate variant ofthe Bad protein has an amino acid other than serine at amino acidposition 112 and/or position 136, to provide constitutively reducedphosphorylation.

[0024] Thus, one specific embodiment is a functional apoptosis-enhancingfusion protein capable of binding a target cell, comprising the Badprotein and the diphtheria toxin translocation and receptor bindingdomains, functionally linked to each other. The Bad protein of thisembodiment can also contain a mutation(s) at position 112 and/or 136 tochange the serine residue to some other amino acid, to reducephosphorylation of the protein. One such protein is Bad-DTTR; thenucleotide sequence of this protein is shown in SEQ ID NO: 3, and thecorresponding amino acid sequence in SEQ ID NOs: 3 and 4.

[0025] Also disclosed herein are nucleic acid molecules encodingapoptosis-modifying fusion proteins, for instance the nucleic acidsequences in SEQ ID NOs: 1, 3, and 7, and nucleic acid sequences havingat least 90% sequence identity to these sequences, for instance thoseencoding for proteins containing one or more conservative amino acidsubstitutions. Other nucleic acid sequences may have 95% or 98% sequenceidentity with SEQ ID NO: 1, 3, or 7. Also encompassed are recombinantnucleic acid molecules in which such a nucleic acid sequence is operablylinked to a promoter, vectors containing such a molecule, and transgeniccells comprising such a molecule.

[0026] Methods also are provided for producing functional recombinantapoptosis-modifying fusion proteins capable of binding to a target cell,integrating into or otherwise translocating across the cell membrane,and modifying an apoptotic response of the target cell. Such a proteincan be produced in a prokaryotic or eukaryotic cell, for instance bytransforming or transfecting such a cell with a recombinant nucleic acidmolecule comprising a sequence which encodes a disclosed bispecificfusion protein. Appropriate eukaryotic cells include yeast, algae, plantor animal cells. Such transformed cells can then be cultured underconditions that cause production of the fusion protein, which is thenrecovered through protein purification means. The protein can include amolecular tag, such as a six histidine (hexa-his) tag, to facilitate itsrecovery.

[0027] Protein analogs, derivatives, or mimetics of the disclosedproteins, which retain the ability to target to appropriate target cellsand modify apoptosis in those cells, are also encompassed inembodiments.

[0028] Compositions containing these apoptosis modifying fusionproteins, and analogs, derivatives, or mimetics of these proteins, arefurther aspects of this disclosure. Such compositions may furthercontain a pharmaceutically acceptable carrier, various other medical ortherapeutic agents, and/or additional apoptosis modifying substances.

[0029] Methods for modifying apoptosis in a target cell are alsoencompassed, wherein a sufficient amount of a fusion protein of thecurrent disclosure to modify apoptosis in the target cell is contactedwith a target cell. Modification of apoptosis can be by eitherinhibition or enhancement of an apoptotic response of the target cell.The fusion protein can be administered to the target cell in the form ofa pharmaceutical composition, and can further be administered withvarious medical or therapeutic agents, and/or additional apoptosismodifying substances. Such agents may include, for instance,chemotherapeutic, anti-inflammatory, anti-viral, and antibiotic agents.

[0030] Bcl-x_(L)-DTR, LF_(n)-Bcl-x_(L), or related fusion proteins canbe used to inhibit apoptosis in a target cell by contacting the targetcell with an amount of this protein sufficient to inhibit apoptosis.Alternatively, Bad-DTTR or related fusion proteins can be used toenhance apoptosis in a target cell by contacting the target cell with anamount of this protein sufficient to enhance apoptosis.

[0031] A specific aspect disclosed herein is the method of reducingapoptosis in a subject after transient ischemic neuronal injury, forinstance a spinal cord injury, comprising administering to the subject atherapeutically effective amount of an apoptosis-inhibiting proteinaccording to this disclosure. Examples of such fusion proteins includeBcl-x_(L)-DTR and LF_(n)-Bcl-x_(L). These proteins can be administeredin the form of a pharmaceutical composition, and can be co-administeredwith various medical or therapeutic agents, and/or additional apoptosismodifying substances.

[0032] The foregoing and other features and advantages of the inventionwill become more apparent from the following detailed description ofseveral embodiments, which proceeds with reference to the accompanyingfigures and tables.

BRIEF DESCRIPTION OF THE FIGURES

[0033]FIG. 1 shows the construction, production and bioactivity ofBcl-x_(L)-DTR and Bcl-x_(L) transfected into HeLa cells. FIG. 1A is aschematic representation of construction of Bcl-x_(L)-DTR. FIG. 1B is aWestern blot of the lysates of HeLa cells transiently transfected withBcl-x_(L) (lane b) and Bcl-x_(L)-DTR (lane c). Lane a containsuntransfected cells as a control. A small amount of endogeneousBcl-x_(L) is present in lanes a and c. FIG 1C is a graph that showstransient transfection of Bcl-x_(L) (∘) and Bcl-x_(L)-DTR (⋄) genes intoHeLa cells inhibits apoptotic cell death induced by the addition of STS.Apoptosis in control cells transfected with the vector (pcDNA3) vectoris shown for comparison (□).

[0034]FIG. 2 is a graph that shows the results of a diphtheria toxinreceptor competitive binding assay. Cold competitor proteins [native DT(Δ), Bcl-x_(L)-DTR (▴), Bcl-x_(L) (∘), and DTR ()] were used todisplace I¹²⁵ labeled diphtheria toxin (DT) tracer, and the amount ofbound, labeled tracer was measured. Native DT and the fusion proteinBcl-x_(L)-DTR compete for DT receptor binding in the nanomolarconcentration range.

[0035]FIG. 3 depicts the results of several experiments that demonstratethe apoptosis-inhibiting character of the fusion constructBcl-x_(L)-DTR. Panel A is a graph of a time course of apoptosis inducedby staurosporine (STS). Cells were treated with 0.1 μM STS (∘), 0.1 μMSTS plus 4.8 μM Bcl-x_(L)-DTR protein medium (Δ), or 20 μl of PBS (□).Results are presented as the average number of apoptotic cells per field(magnification 160×). For each point, at least 5 fields were counted ineach of at least 3 wells. FIG. 3B is a SDS-PAGE gel that shows thatBcl-x_(L)-DTR prevents PARP cleavage. Lane a contains control HeLa cellsnot incubated with STS (uninduced cells); Lane b, HeLa cells treatedwith STS plus 1 μM Bcl-x_(L)-DTR protein; Lane c, HeLa cells treatedwith STS plus 1.48 μM Bcl-x_(L)-DTR protein; and Lane d, HeLa cellstreated with STS and no fusion protein.

[0036]FIG. 4 shows that Bcl-x_(L)-DTR inhibits of apoptosis induced byγ-radiation, but not that induced by α-Fas antibody. FIG. 4A is a graphshowing that the addition of Bcl-x_(L)-DTR prior to irradiation ofJurkat cells reduces apoptotic death in response to γ-radiation. Controlcells were not irradiated and not treated with Bcl-x_(L)-DTR. FIG. 4B isa graph that shows that, in Jurkat cells, Bcl-x_(L)-DTR had littleinhibitory effect on apoptosis induced by anti-Fas antibody. Controlcells were treated with PBS and no anti-Fas antibody.

[0037]FIG. 5 shows that Bcl-x_(L)-DTR inhibits apoptosis induced bypoliovirus.

[0038]FIG. 6 is a graph showing the time course of viability of cellstreated with Bad-DTTR.

[0039]FIG. 7 shows the results of experiments that demonstrate thatBad-DTTR combined with STS triggers massive cell death. FIG. 7A is agraph quantifying cell death after treatment of U251 MG cells withvarious combinations of STS and Bad-DTTR. Apoptosis is most enhancedwhen cells are treated with 0.1 μM STS plus 0.65 μM Bad-DTTR, and cellsbegin to die about 12 hours after treatment. In the experiment depictedin FIG. 7B, the use of 1 μM STS in combination with variousconcentrations of Bad-DTTR cause an earlier onset of apoptosis in U251MG cells. Key: □=PBS; ⋄=0.1 μM STS; ∘=0.65 μM Bad-DTTR; Δ=0.065 μMBad-DTTR;

=0.1 μM STS +0.65 μM Bad-DTTR; ⊖=0.1 μM STS +0.065 μM Bad-DTTR.

[0040]FIG. 8 is a schematic diagram of the chimera LF_(n)-Bcl-x_(L). Thefusion gene, LF_(n)-Bcl-x_(L), was inserted into the vector, pET15b,yielding a histidine tag sequence at the N terminus of theLF_(n)-Bcl-x_(L) gene.

[0041]FIG. 9 is a graph showing the time course of apoptosis induced bySTS in J774 cells, with or without LF_(n)-Bcl-x_(L) protein. J774 cellsat 3×10⁴/ cm² were treated with 0.1 μM staurosporine alone, 0.1 μMstaurosporine along with LF_(n)-Bcl-x_(L) (28 μg/ml) plus PA (33 μg/ml),or with PBS alone. The apoptotic and living cells were stained withHoechst 33342 and counted at the indicated times, and the data werecalculated as reported (Liu et al., Proc. Natl. Acad. Sci. USA 96:9563-9567, 1999).

[0042]FIG. 10 is a bar graph showing the effect of LF_(n)-Bcl-x_(L)against J774 treated with STS. J774 cells at 10⁴/cm² were treated withPBS, 0.1 μM staurosporine alone, 0.1 μM staurosporine along with LF_(n)(28 μg/ml), 0.1 μM staurosporine along with Bcl-x_(L) (28 μg/ml), 0.1 μMstaurosporine along with LF_(n)-Bcl-x_(L) (28 μg/ml), 0.1 μMstaurosporine along with LF_(n)-Bcl-x_(L) (28 μg/ml) plus PA (33 μg/ml),0.1 μM staurosporine along with PA (33 μg/ml) and 0.1 μM staurosporinealong with LF_(n) (28 μg/ml) plus PA (33 μg/ml). The apoptotic andliving cells were stained with Hoechst 33342 48 hours later and counted,and the data were calculated as for FIG. 9.

[0043]FIG. 11 is a bar graph showing the effect of LF_(n)-Bcl-x_(L)against Jurkat cells treated with STS. Jurkat cells at 10⁵/ml weretreated with 0.1 μM staurosporine alone, 0.1 μM staurosporine along withLF_(n)-Bcl-x_(L) (28 μg/ml) plus PA (33 μg/ml) or with PBS. Theapoptotic and living cells were stained with Hoechest 33342 21 hourslater and counted, and the data were calculated as for FIG. 9.

[0044]FIG. 12 is a bar graph showing that the fusion proteinLF_(n)-Bcl-x_(L) prevents apoptosis by in neonatal rat retinal ganglioncells 24 hours after optic nerve section. The apoptotic and living cellsin retinal ganglion layers were counted 24 hours after optic nervesection immediately followed by the injection of PBS or the indicatedprotein(s). The percentage of apoptotic cells versus total retinalganglion cells per retina is represented.

SEQUENCE LISTING

[0045] The nucleic and amino acid sequences listed in the accompanyingsequence listing are shown using standard letter abbreviations fornucleotide bases, and three letter code for amino acids. Only one strandof each nucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand.

[0046] SEQ ID NO: 1 shows the DNA coding sequence and correspondingamino acid sequence of Bcl-x_(L)-DTR.

[0047] SEQ ID NO: 2 shows the amino acid sequence of Bcl-x_(L)-DTR.

[0048] SEQ ID NO: 3 shows the DNA coding sequence and correspondingamino acid sequence of Bad-DTTR.

[0049] SEQ ID NO: 4 shows the amino acid sequence of Bad-DTTR.

[0050] SEQ ID NO: 5 shows the nucleotide sequence of the linker used tolink Bcl-x_(L) to DTR in the fusion construct Bcl-x_(L)-DTR.

[0051] SEQ ID NO: 6 shows the amino acid sequence of the linker used tolink Bcl-x_(L) to DTR to form Bcl-x_(L)-DTR.

[0052] SEQ ID NO: 7 shows the DNA coding sequence and correspondingamino acid sequence of LF_(n)-Bcl-x_(L).

[0053] SEQ ID NO: 8 shows the amino acid sequence of LF_(n)-Bcl-x_(L).

DETAILED DESCRIPTION OF THE INVENTION I. Abbreviations and Definitions

[0054] A. Abbreviations

[0055] DT: diphtheria toxin

[0056] DTR: diphtheria toxin receptor binding domain

[0057] DTT: diphtheria toxin translocation domain

[0058] DTTR: diphtheria toxin translocation and receptor binding domainsE. coli: Escherichia coli

[0059] EF: anthrax edema factor

[0060] LF: anthrax lethal factor

[0061] LF_(n): first 255 residues of anthrax lethal factor

[0062] moi: multiplicity of infection

[0063] PA: anthrax protective antigen

[0064] PCR: polymerase chain reaction

[0065] RE: restriction endonuclease

[0066] SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gelelectrophoresis

[0067] STS: staurosporine

[0068] TdT: terminal deoxyribonucleotidyl transferase

[0069] TUNEL: TdT-dependent dUTP-biotin nick end labeling

[0070] B. Definitions

[0071] Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Lewin, Genes V published by Oxford University Press, 1994(ISBN 0-19-854287-9); Kendrew et al., (eds.), The Encyclopedia ofMolecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8). The nomenclature for DNAbases and the three-letter code for amino acid residues, as set forth at37 CFR § 1.822, are used herein.

[0072] In order to facilitate review of the various embodiments of theinvention, the following definitions of terms are provided. Thesedefinitions are not intended to limit such terms to a scope narrowerthan would be known to a person of ordinary skill in the field.

[0073] Animal: Living multi-cellular vertebrate organisms, a categorythat includes, for example, mammals and birds. The term mammal includesboth human and non-human mammals. Similarly, the term “subject” includesboth human and veterinary subjects.

[0074] Apoptosis-modifying ability: A protein has apoptosis-modifyingability if it is capable of modifying apoptosis in a cell. This abilityis usually measurable, either in vivo or in vitro, using any one ofmyriad apoptosis assays. The art is replete with methods for measuringapoptosis. Appropriate techniques include dye exclusion (e.g. Hoechstdye No. 33342), assaying for caspase activity, and TUNEL-staining. Thespecific ability of a fusion protein to modify the apoptotic response ofa cell to various apoptosis-inducing stimuli can be determined byrunning standard apoptosis assays in the absence of or presence ofvarious concentrations of the fusion proteins. The results of the assayare then compared, and can be reported for instance by presenting thepercentage of apoptosis that occurs in the presence of the fusionprotein.

[0075] The invention also includes analogs, derivatives or mimetics ofthe disclosed fusion proteins, and which have apoptosis-modifyingability. Such molecules can be screened for apoptosis-modifying abilityby assaying a protein similar to the disclosed fusion protein, in thatit has one or more conservative amino acid substitutions or shortin-frame deletions or insertions, or analogs, derivatives or mimeticsthereof, and determining whether the similar protein, analog, derivativeor mimetic provides modification of apoptosis in a desired target cell.The apoptosis-modifying ability and target cell binding affinity ofthese derivative compounds can be measured by any known means, includingthose discussed in this application.

[0076] Apoptosis-modifying fusion protein: Proteins that have at leasttwo domains fused together, at least one domain comprising a cellbinding region capable of targeting the fusion protein to a target cell(the targeting or cell-binding domain), and at least one domain capableof modifying apoptosis in the target cell (the apoptosis-modifyingdomain). The apoptosis-modifying fusion proteins of the currentinvention are further characterized by their ability to integrate intoor otherwise cross a cellular membrane of the target cell when deliveredextracellularly. An apoptosis-modifying fusion protein is consideredfunctional if it targets to the correct target cell, and modifies anapoptotic response of that cell.

[0077] In general, the two domains of the disclosed fusions aregenetically fused together, in that nucleic acid molecules that encodeeach protein domain are functionally linked together, for instancedirectly or through the use of a linker oligonucleotide, therebyproducing a single fusion-encoding nucleic acid molecule. The translatedproduct of such a fusion-encoding nucleic acid molecule is theapoptosis-modifying fusion protein.

[0078] Apoptosis-modifying fusion proteins can be labeled according tohow they influence apoptosis in the target cell. For instance, anapoptosis-modifying fusion protein according to the current inventionthat inhibits apoptosis in the target cell can be referred to as anapoptosis-inhibiting fusion protein (e.g., Bcl-x_(L)-DTR andLF_(n)-Bcl-x_(L)). Likewise, if the fusion protein enhances apoptosis inthe target cell, it can be referred to as an apoptosis-enhancing fusionprotein (e.g., Bad-DTTR). Specific apoptosis-modifying fusion proteinsare usually named for the proteins from which domains are taken to formthe fusion, or from the domains actually used. For instance,“Bcl-x_(L)-DTR” (SEQ ID NOs: 1 and 2) consists of the entire Bcl-x_(L)protein fused in frame to the receptor-binding domain of diphtheriatoxin (DTR) via a short linker.

[0079] A Bcl-2 protein: A Bcl-2 protein is a protein from the Bcl-2family of proteins and includes those proteins related to Bcl-2 bysequence homology, which affect apoptosis. By way of example, the familyincludes Bcl-2, Bcl-x (both the long and short forms), Bax, and Bad.Additional members of the Bcl-2 family of proteins are known (Adams andCory, Science 281:1322-1326, 1998).

[0080] Molecules that are derived from proteins of the Bcl-2 familyinclude fragments of such proteins (e.g., fragments of Bcl-x_(L) orBad), generated either by chemical (e.g., enzymatic) digestion orgenetic engineering means. Such fragments may comprise nearly all of thenative protein, with one or a few amino acids being genetically orchemically removed from the amino or carboxy terminal end of theprotein, or genetically removed from an internal region of the sequence.

[0081] Derived molecules, or derived from: The term “X-derivedmolecules” or “derived from X,” where X is a protein also encompassesanalogs (non-protein organic molecules), derivatives (chemicallyfunctionalized protein molecules obtained starting with the disclosedprotein sequences) or mimetics (three-dimensionally similar chemicals)of the native protein structure, as well as proteins sequence variantsor genetic alleles, that maintain biological functionality. Where thederived molecule is used as the targeting domain of anapoptosis-modifying fusion protein, the biological functionalitymaintained is the ability to target to fusion protein to the desiredtarget cell. Likewise, where the derived molecule is used as theapoptosis-modifying domain of the fusion, the functionality maintainedis the ability to affect apoptosis in the target cell. Each of thesefunctionalities can be measured in various ways, including specificprotein binding and apoptosis assays, respectively.

[0082] Injectable composition: A pharmaceutically acceptable fluidcomposition comprising at least one active ingredient, e.g., anapoptosis-modifying fusion protein. The active ingredient is usuallydissolved or suspended in a physiologically acceptable carrier, and thecomposition can additionally comprise minor amounts of one or morenon-toxic auxiliary substances, such as emulsifying agents,preservatives, and pH buffering agents and the like. Such injectablecompositions that are useful for use with the fusion proteins of thisinvention are conventional; appropriate formulations are well known inthe art.

[0083] Isolated: An “isolated” biological component (such as a nucleicacid molecule, protein or organelle) has been substantially separated orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, i.e., otherchromosomal and extra-chromosomal DNA and RNA, proteins and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinantexpression in a host cell as well as chemically synthesized nucleicacids.

[0084] Linker: A peptide, usually between two and 150 amino acidresidues in length, which serves to join two protein domains in amulti-domain fusion protein. Peptide linkers are generally encoded forby a corresponding oligonucleotide linker. This can be geneticallyfused, in frame, between the nucleotides that encode the domains of afusion protein.

[0085] Oligonucleotide: A linear polynucleotide sequence of between sixand 300 nucleotide bases in length.

[0086] Operably linked: A first nucleic acid sequence is operably linkedwith a second nucleic acid sequence when the first nucleic acid sequenceis placed in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

[0087] Parenteral: Administered outside of the intestine, e.g., not viathe alimentary tract. Generally, parenteral formulations are those thatwill be administered through any possible mode except ingestion. Thisterm especially refers to injections, whether administeredintravenously, intrathecally, intramuscularly, intraperitoneally, orsubcutaneously, and various surface applications including intranasal,intradermal, and topical application, for instance.

[0088] Pharmaceutically acceptable carriers: The pharmaceuticallyacceptable carriers useful in this invention are conventional. Martin,Remington 's Pharmaceutical Sciences, published by Mack Publishing Co.,Easton, Pa., 15th Edition, 1975, describes compositions and formulationssuitable for pharmaceutical delivery of the fusion proteins hereindisclosed.

[0089] In general, the nature of the carrier will depend on theparticular mode of administration being employed. For instance,parenteral formulations usually comprise injectable fluids that includepharmaceutically and physiologically acceptable fluids such as water,physiological saline, balanced salt solutions, aqueous dextrose,glycerol or the like as a vehicle. For solid compositions (e.g., powder,pill, tablet, or capsule forms), conventional non-toxic solid carrierscan include, for example, pharmaceutical grades of mannitol, lactose,starch, or magnesium stearate. In addition to biologically-neutralcarriers, pharmaceutical compositions to be administered can containminor amounts of non-toxic auxiliary substances, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like,for example sodium acetate or sorbitan monolaurate.

[0090] Purified: The term purified does not require absolute purity;rather, it is intended as a relative term. Thus, for example, a purifiedfusion protein preparation is one in which the fusion protein is moreenriched than the protein is in its generative environment, for instancewithin a cell or in a biochemical reaction chamber. Preferably, apreparation of fusion protein is purified such that the fusion proteinrepresents at least 50% of the total protein content of the preparation.More purified preparations will have fusion protein that represents atleast 60%, 70%, 80% or 90% of the total protein content.

[0091] Recombinant: A recombinant nucleic acid molecule is one that hasa sequence that is not naturally occurring or has a sequence that ismade by an artificial combination of two otherwise separated segments ofsequence. This artificial combination can be accomplished by chemicalsynthesis or, more commonly, by the artificial manipulation of isolatedsegments of nucleic acids, e.g., by genetic engineering techniques.

[0092] Similarly, a recombinant protein is one encoded for by arecombinant nucleic acid molecule.

[0093] Sequence identity: The similarity between two nucleic acidsequences, or two amino acid sequences is expressed in terms of thesimilarity between the sequences, otherwise referred to as sequenceidentity. Sequence identity is frequently measured in terms ofpercentage identity (or similarity or homology); the higher thepercentage, the more similar the two sequences are. Homologs of theapoptosis-modifying fusion protein will possess a relatively high degreeof sequence identity when aligned using standard methods. For instance,encoding sequences encompassed in the current invention include thosethat share about 90% sequence identity with SEQ ID NO: 1 and NO: 3.

[0094] Methods of alignment of sequences for comparison are well knownin the art. Various programs and alignment algorithms are described in:Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch,J. Mol. Biol. 48:443, 1970; Pearson and Lipman, PNAS. USA 85:2444, 1988;Higgins and Sharp, Gene, 73:237-244, 1988; Higgins and Sharp, CABIOS5:151-153, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huanget al., Comp. Appls Biosci. 8:155-65, 1992; and Pearson et al., Meth.Mol. Biol. 24:307-31, 1994. Altschul et al., Nature Genet. 6:119-29,1994, presents a detailed consideration of sequence alignment methodsand homology calculations.

[0095] The alignment tools ALIGN (Myers and Miller, CABIOS 4:11- 17,1989) or LFASTA (Pearson and Lipman, PNAS. USA 85:2444, 1988) may beused to perform sequence comparisons (Internet Program© 1996, W. R.Pearson and the University of Virginia, “fasta20u63” version 2.0u63,release date December 1996). ALIGN compares entire sequences against oneanother, while LFASTA compares regions of local similarity. Thesealignment tools and their respective tutorials are available on theInternet at the NCSA web-site.

[0096] For comparisons of amino acid sequences of greater than about 30amino acids, the Blast 2 sequences function is employed using thedefault BLOSUM62 matrix set to default parameters, (gap existence costof 11, and a per residue gap cost of 1). When aligning short peptides(fewer than around 30 amino acids), the alignment should be performedusing the Blast 2 sequences function, employing the PAM30 matrix set todefault parameters (open gap 9, extension gap 1 penalties). Proteinswith even greater similarity to the reference sequences will showincreasing percentage identities when assessed by this method, such asat least 90%, at least 92%, at least 94%, at least 95%, at least 97%, atleast 98%, or at least 99% sequence identity.

[0097] An alternative indication that two nucleic acid molecules areclosely related is that the two molecules hybridize to each other understringent conditions. Stringent conditions are sequence-dependent andare different under different environmental parameters. Generally,stringent conditions are selected to be about 5° C. to 20° C. lower thanthe thermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. Conditions for nucleic acid hybridization andcalculation of stringencies can be found in Sambrook et al., InMolecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989;and Tijssen, Laboratory Techniques in Biochemistry and Molecular BiologyPart I, Ch. 2, Elsevier, N.Y., 1993. Nucleic acid molecules thathybridize to the disclosed apoptosis-modifying fusion protein sequencesunder stringent conditions will typically hybridize to a probe (based onthe entire fusion protein encoding sequence, an entire domain, or otherselected portions of the encoding sequence) under wash conditions of0.2×SSC, 0.1% SDS at 65° C.

[0098] Nucleic acid sequences that do not show a high degree of identitymay nevertheless encode similar amino acid sequences, due to thedegeneracy of the genetic code. It is understood that changes in nucleicacid sequence can be made using this degeneracy to produce multiplenucleic acid sequences that encode substantially the same protein.

[0099] Specific binding agent: An agent that binds substantially only toa defined target. Thus a Bcl-x_(L)-DTR-specific binding agent bindssubstantially only the Bcl-x_(L)-DTR protein in a specific preparation.As used herein, the term “Bcl-x_(L)-DTR-specific binding agent” includesBcl-x_(L)-DTR antibodies and other agents that bind substantially onlyto a Bcl-x_(L)-DTR protein in that preparation.

[0100] Anti-Bcl-x_(L)-DTR antibodies may be produced using standardprocedures described in a number of texts, including Harlow and Lane(Using Antibodies, A Laboratory Manual, CSHL, New York, 1999, ISBN0-87969-544-7). The determination that a particular agent bindssubstantially only to Bcl-x_(L)-DTR protein may readily be made by usingor adapting routine procedures. One suitable in vitro assay makes use ofthe Western blotting procedure (described in many standard texts,including Harlow and Lane, 1999). Western blotting may be used todetermine that a given protein binding agent, such as ananti-Bcl-x_(L)-DTR monoclonal antibody, binds substantially only to theBcl-x_(L)-DTR protein.

[0101] Alternately, because the disclosed apoptosis-modifying proteinsare fusion proteins, they can be detected using antibodies to one or theprotein domains used in their construction. For instance, fusionscontaining Bcl-x_(L) can be detected using the monoclonal antibody 2H12(Hsu and Youle, J. Biol. Chem. 272:13829-13834, 1997; now available fromNeo Markers, Union City, Calif., clone #2H121-3) or other professionallyavailable antibody preparations, for instance, polyclonalanti-Bcl-x_(L)/x_(s) #06-851 from Upstate Biotechnology, Lake Placid,N.Y.; polyclonal rabbit anti-Bcl-x_(L) #65189E from PharMingen, SanDiego, Calif.; and rabbit polyclonal (#B22630-050/B22630-150) or mousemonoclonal (B61220-050/B61220-150) anti-Bcl-x_(L) from TransductionLaboratories, Lexington, Ky.). Antibodies that recognize diphtheriatoxin are, for instance, available from the Centers for Disease Control,Atlanta, Ga.

[0102] Shorter fragments of antibodies can also serve as specificbinding agents. For instance, FAbs, Fvs, and single-chain Fvs (SCFvs)that bind to Bcl-x_(L)-DTR would be Bcl-x_(L)-DTR-specific bindingagents.

[0103] Target cell binding affinity: The physical interaction between atarget cell and an apoptosis-modifying fusion protein as disclosed inthis invention can be examined by various methods. Alternatively, theability of fusion protein to compete for binding to its target cell witheither native targeting domain or antibody that recognizes the targetingdomain binding site on the target cell can be measured. This allows thecalculation of relative binding affinities through standard techniques.

[0104] Therapeutically effective amount of an apoptosis-modifying fusionprotein: A quantity of apoptosis-modifying fusion protein sufficient toachieve a desired effect in a subject being treated. For instance, thiscan be the amount necessary to measurably inhibit or enhance apoptosisin a target cell.

[0105] An effective amount of apoptosis-modifying fusion protein may beadministered in a single dose, or in several doses, for example daily,during a course of treatment. However, the effective amount of fusionprotein will be dependent on the fusion protein applied, the subjectbeing treated, the severity and type of the affliction, and the mannerof administration of the fusion protein. For example, a therapeuticallyeffective amount of fusion protein can vary from about 0.01 mg/kg bodyweight to about 1 g/kg body weight.

[0106] The fusion proteins disclosed in the present invention have equalapplication in medical and veterinary settings. Therefore, the generalterm “subject being treated” is understood to include all animals (e.g.,humans, apes, dogs, cats, horses, and cows), and particularly mammals,that are or may suffer from a chronic or acute condition or injury thatcauses apoptosis, or a lack thereof, susceptible to modification usingmolecules of the current invention.

[0107] Transformed: A transformed cell is a cell into which has beenintroduced a nucleic acid molecule by molecular biology techniques. Asused herein, the term transformation encompasses all techniques by whicha nucleic acid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration.

[0108] Transgenic cell: A transgenic cell is one that has beentransformed with a recombinant nucleic acid molecule.

[0109] Vector: A nucleic acid molecule as introduced into a host cell,thereby producing a transformed host cell. A vector may include nucleicacid sequences that permit it to replicate in a host cell, such as anorigin of replication. A vector may also include one or more selectablemarker genes and other genetic elements known in the art.

II. Construction, Expression, and Purification of Apoptosis-ModifyingFusion Proteins.

[0110] A. Selection of component Domains.

[0111] This invention provides generally an apoptosis-modifying fusionprotein that binds to a target cell, translocates across or otherwiseintegrates into the membrane(s) of the target cell, and modifies anapoptotic response of the target cell. As such, any target cell in whichit is desirous to modify (either inhibit or enhance) apoptosis is anappropriate target for a bispecific fusion protein. The choice ofappropriate protein binding domain for incorporation into the disclosedapoptosis-modifying fusion protein will be dictated by the target cellor cell population chosen. Examples of targeting domains include, forinstance, nontoxic cell binding domains or components of bacterialtoxins (such as diphtheria toxin or anthrax toxin), growth factors (suchas epidermal growth factor), monoclonal antibodies, cytokines, and soforth, as well as targeting competent variants and fragments thereof.

[0112] The choice of appropriate Bcl-2 family member-derivedapoptosis-modifying domain will depend on the manner in which the targetcell's response to apoptosis is to be modified. Where apoptosis is to beinhibited by the resultant fusion protein, anti-death members of theBcl-2 protein family are appropriate sources for apoptosis-modifyingdomains. One such fusion protein is Bcl-x_(L)-DTR, which employs thelong form of Bcl-x, Bcl-x_(L), as the apoptosis-modifying domain.Alternately, where enhancement of apoptosis is desired, pro-deathmembers of the Bcl-2 family of proteins will be appropriate. Forinstance, Bad-DTTR employs the pro-death protein Bad as itsapoptosis-modifying domain.

[0113] Translocation of the apoptosis-modifying fusion protein into thetarget cell is important. A translocation domain may be included in thefusion protein as a separate, third domain. This could be supplied froma third protein, unrelated to the cell-binding and apoptosis-modifyingdomains, or be a translocation domain of one of these proteins (e.g.,the diphtheria toxin translocation (DTT) domain used in Bad-DTTR). TheDTT domain contains several hydrophobic and amphipathic alpha helicesand, after insertion into cell membranes, creates voltage dependent ionchannels (Kagan et al., Proc Natl Acad Sci USA 78:4950-4954, 1981;Donovan et al., Proc Natl Acad Sci USA 78:172-176, 1981).

[0114] Alternately, the translocation function can be provided throughthe use of a cell-binding domain or apoptosis-modifying domain thatconfers the additional functionality of membrane translocation orintegration. This is true in Bcl-x_(L)-DTR, wherein Bcl-x_(L) providesboth the apoptosis-modifying ability and translocation into the cell.

[0115] B. Assembly

[0116] The construction of fusion proteins from domains of knownproteins is well known. In general, a nucleic acid molecule that encodesthe desired protein domains are joined using standard geneticengineering techniques to create a single, operably linked fusionoligonucleotide. Appropriate molecular biological techniques may befound in Sambrook et al., In Molecular Cloning: A Laboratory Manual,Cold Spring Harbor, N.Y., 1989. Specific examples of geneticallyengineered multi-domain proteins, including those joined by variouslinkers, can be found in the following patent documents:

[0117] U.S. Pat. No. 5,834,209 to Korsmeyer;

[0118] U.S. Pat. No. 5,821,082 to Chinnadurai;

[0119] U.S. Pat. No. 5,696,237 to FitzGerald et al.;

[0120] U.S. Pat. No. 5,668,255 to Murphy;

[0121] U.S. Pat. No. 5,587,455 to Berger et al.;

[0122] WO 98/17682 to Korsmeyer; and

[0123] WO 98/12328 to Home et al.

[0124] It will usually be convenient to generate various controlmolecules for comparison to an apoptosis-modifying fusion protein, inorder to measure the specificity of the apoptosis modification providedby each fusion protein. Appropriate control molecules may include one ormore of the native proteins used in construction of the fusion, orfragments or mutants thereof.

[0125] C. Expression

[0126] One skilled in the art will understand that there are myriad waysto express a recombinant protein such that it can subsequently bepurified. In general, an expression vector carrying the nucleic acidsequence that encodes the desired protein will be transformed into amicroorganism for expression. Such microorganisms can be prokaryotic(bacteria) or eukaryotic (e.g., yeast). One appropriate species ofbacteria is Escherichia coli (E. coli), which has been used extensivelyas a laboratory experimental expression system. A eukaryotic expressionsystem will be preferred where the protein of interest requireseukaryote-specific post-translational modifications such asglycosylation. Also, protein can be expressed using a viral (e.g.,vaccinia) based expression system.

[0127] Protein can also be expressed in animal cell tissue culture, andsuch a system will be appropriate where animal-specific proteinmodifications are desirable or required in the recombinant protein.

[0128] The expression vector can include a sequence encoding a synthesistargeting peptide, positioned in such a way as to be fused to the codingsequence of the apoptosis-modifying fusion protein. This allows theapoptosis-modifying fusion protein to be targeted to specificsub-cellular or extra-cellular locations. Various appropriateprokaryotic and eukaryotic targeting peptides, and nucleic acidmolecules encoding such, are well known to one of ordinary skill in theart. In a prokaryotic expression system, a signal sequence can be usedto secrete the newly synthesized protein. In a eukaryotic expressionsystem, the targeting peptide would specify targeting of the hybridprotein to one or more specific sub-cellular compartments, or to besecreted from the cell, depending on which peptide is chosen. Throughthe use of a eukaryotic secretion signal sequence, theapoptosis-modifying fusion protein can be expressed in a transgenicanimal (for instance a cow, pig, or sheep) in such a manner that theprotein is secreted into the milk of the animal.

[0129] Vectors suitable for stable transformation of culturable cellsare also well known. Typically, such vectors include a multiple-cloningsite suitable for inserting a cloned nucleic acid molecule, such that itwill be under the transcriptional control of 5′ and 3′ regulatorysequences. In addition, transformation vectors include one or moreselectable markers; for bacterial transformation this is often anantibiotic resistance gene. Such transformation vectors typically alsocontain a promoter regulatory region (e.g., a regulatory regioncontrolling inducible or constitutive expression), a transcriptioninitiation start site, a ribosome binding site, an RNA processingsignal, and a transcription termination site, each functionally arrangedin relation to the multiple-cloning site. For production of largeamounts of recombinant proteins, an inducible promoter is preferred.This permits selective production of the recombinant protein, and allowsboth higher levels of production than constitutive promoters, andenables the production of recombinant proteins that may be toxic to theexpressing cell if expressed constitutively.

[0130] In addition to these general guidelines, proteinexpression/purification kits are produced commercially. See, forinstance, the QIAexpress™ expression system from QIAGEN (Chatsworth,Calif.) and various expression systems provided by INVITROGEN (Carlsbad,Calif.). Depending on the details provided by the manufactures, suchkits can be used for production and purification of the disclosedapoptosis-modifying fusion proteins.

[0131] D. Purification

[0132] One skilled in the art will understand that there are myriad waysto purify recombinant polypeptides, and such typical methods of proteinpurification may be used to purify the disclosed apoptosis-modifyingfusion proteins. Such methods include, for instance, proteinchromatographic methods including ion exchange, gel filtration, HPLC,monoclonal antibody affinity chromatography and isolation of insolubleprotein inclusion bodies after over production. In addition,purification affinity-tags, for instance a six-histidine sequence, maybe recombinantly fused to the protein and used to facilitate polypeptidepurification. A specific proteolytic site, for instance athrombin-specific digestion site, can be engineered into the proteinbetween the tag and the fusion itself to facilitate removal of the tagafter purification.

[0133] Commercially produced protein expression/purification kitsprovide tailored protocols for the purification of proteins made usingeach system. See, for instance, the QIAexpress™ expression system fromQIAGEN (Chatsworth, Calif.) and various expression systems provided byINVITROGEN (Carlsbad, Calif.). Where a commercial kit is employed toproduce a bispecific fusion protein, the manufacturer's purificationprotocol is a preferred protocol for purification of that protein. Forinstance, proteins expressed with an amino-terminal hexa-histidine tagcan be purified by binding to nickel-nitrilotriacetic acid (Ni-NTA)metal affinity chromatography matrix (The QIAexpressionist, QIAGEN,1997).

[0134] Alternately, the binding specificities of thecell-binding/targeting domain of the disclosed apoptosis-modifyingprotein may be exploited to facilitate specific purification of theproteins. A preferred method of performing such specific purificationwould be column chromatography using column resin to which the targetcell surface receptor, or an appropriate epitope or fragment or domainof the target molecule, has been attached.

[0135] If the apoptosis-modifying fusion protein is produced in asecreted form, e.g. secreted into the milk of a transgenic animal,purification will be from the secreted fluid. Alternately, purificationmay be unnecessary if it is appropriate to apply the fusion proteindirectly to the subject in the secreted fluid (e.g. milk).

III. Variation of a Bispecific Fusion Protein

[0136] A. Sequence Variants

[0137] The binding and apoptosis-modifying characteristics of theapoptosis-modifying fusion proteins disclosed herein lies not in theprecise amino acid sequence, but rather in the three-dimensionalstructure inherent in the amino acid sequences encoded by the DNAsequences. It is possible to recreate the functional characteristics ofany of these proteins or protein domains of this invention by recreatingthe three-dimensional structure, without necessarily recreating theexact amino acid sequence. This can be achieved by designing a nucleicacid sequence that encodes for the three-dimensional structure, butwhich differs, for instance by reason of the redundancy of the geneticcode. Similarly, the DNA sequence may also be varied, while stillproducing a functional apoptosis-modifying fusion protein.

[0138] Variant apoptosis-modifying fusion proteins include proteins thatdiffer in amino acid sequence from the disclosed sequence but that sharestructurally significant sequence homology with any of the providedproteins. Variation can occur in any single domain of the fusion protein(e.g., the binding or apoptosis-modifying domain, or, where appropriate,the linker). Variation can also occur in more than one of such domainsin any particular variant protein. Such variants may be produced bymanipulating the nucleotide sequence of, for instance, aBcl-x_(L)-encoding sequence, using standard procedures, such assite-directed mutagenesis or PCR. The simplest modifications involve thesubstitution of one or more amino acids for amino acids having similarbiochemical properties. These so-called conservative substitutions arelikely to have minimal impact on the activity of the resultant protein,especially when made outside of the binding site or active site of therespective domain. The regions or sub-domains of DTR that are essentialto targeted cell binding are known in the art (see, Choe et al., Nature357:216-222, 1992; Parker and Pattus, TIBS 18:391-395, 1993). Regions orsub-domains of Bcl-2 proteins responsible for apoptosis modification areunder intense study; much of this work is reviewed in Adams and Cory,Science 281:1322-1326.

[0139] Table 1 shows amino acids that may be substituted for an originalamino acid in a protein, and which are regarded as conservativesubstitutions. TABLE 1 Original Residue Conservative Substitutions Alaser Arg lys Asn gln; his Asp glu Cys ser Gln asn Glu asp Gly pro Hisasn; gln Ile leu; val Leu ile; val Lys arg; gln; glu Met leu; ile Phemet; leu; tyr Ser thr Thr ser Trp tyr Tyr trp; phe Val ile; leu

[0140] More substantial changes in protein structure may be obtained byselecting amino acid substitutions that are less conservative than thoselisted in Table 1. Such changes include changing residues that differmore significantly in their effect on maintaining polypeptide backbonestructure (e.g., sheet or helical conformation) near the substitution,charge or hydrophobicity of the molecule at the target site, or bulk ofa specific side chain. The following substitutions are generallyexpected to produce the greatest changes in protein properties: (a) ahydrophilic residue (e.g., seryl or threonyl) is substituted for (or by)a hydrophobic residue (e.g., leucyl, isoleucyl, phenylalanyl, valyl oralanyl); (b) a cysteine or proline is substituted for (or by) any otherresidue; (c) a residue having an electropositive side chain (e.g.,lysyl, arginyl, or histadyl) is substituted for (or by) anelectronegative residue (e.g., glutamyl or aspartyl); or (d) a residuehaving a bulky side chain (e.g., phenylalanine) is substituted for (orby) one lacking a side chain (e.g., glycine).

[0141] Variant binding domain, apoptosis-modifying domain, or fusionprotein-encoding sequences may be produced by standard DNA mutagenesistechniques, for example, M13 primer mutagenesis. Details of thesetechniques are provided in Sambrook et al., In Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, N.Y., 1989, Ch. 15. By the use ofsuch techniques, variants may be created which differ in minor ways fromthe apoptosis-modifying fusion protein-encoding sequences disclosed. DNAmolecules and nucleotide sequences which are derivatives of thosespecifically disclosed herein and that differ from those disclosed bythe deletion, addition, or substitution of nucleotides while stillencoding a protein that binds to a target cell, translocates orotherwise integrates into the target cell membrane(s), and therebymodifies an apoptotic response in the target cell, are comprehended bythis invention. In their most simple form, such variants may differ fromthe disclosed sequences by alteration of the coding region to fit thecodon usage bias of the particular organism into which the molecule isto be introduced.

[0142] Alternatively, the coding region may be altered by takingadvantage of the degeneracy of the genetic code to alter the codingsequence such that, while the nucleotide sequence is substantiallyaltered, it nevertheless encodes a protein having an amino acid sequencesubstantially similar to the disclosed fusion sequences. For example,the 57th amino acid residue of the Bcl-x_(L)-DTR protein is alanine. Thenucleotide codon triplet GCC encodes this alanine residue. Because ofthe degeneracy of the genetic code, three other nucleotide codontriplets—(GCG, GCT and GCA)—also code for alanine. Thus, the nucleotidesequence of the disclosed Bcl-x_(L)-DTR encoding sequence could bechanged at this position to any of these three alternative codonswithout affecting the amino acid composition or characteristics of theencoded protein. Based upon the degeneracy of the genetic code, variantDNA molecules may be derived from the cDNA and gene sequences disclosedherein using standard DNA mutagenesis techniques as described above, orby synthesis of DNA sequences. Thus, this invention also encompassesnucleic acid sequences which encode an apoptosis-modifying fusionprotein, but which vary from the disclosed nucleic acid sequences byvirtue of the degeneracy of the genetic code. Apoptosis assays,including those discussed herein, can be used to determine the abilityof the resultant variant protein to modify apoptosis.

[0143] B. Peptide Modifications

[0144] The present invention includes biologically active molecules thatmimic the action of the apoptosis-modifying fusion proteins of thepresent invention, and specifically modify apoptosis in a target cell.The proteins of the invention include synthetic versions ofnaturally-occurring proteins described herein, as well as analogues(non-peptide organic molecules), derivatives (chemically functionalizedprotein molecules obtained starting with the disclosed peptidesequences) and variants (homologs) of these proteins that specificallybind to a chosen target cell and modify apoptosis in that target cell.Each protein of the invention is comprised of a sequence of amino acids,which may be either L- and/or D- amino acids, naturally occurring andotherwise.

[0145] Proteins may be modified by a variety of chemical techniques toproduce derivatives having essentially the same activity as theunmodified proteins, and optionally having other desirable properties.For example, carboxylic acid groups of the protein, whethercarboxyl-terminal or side chain, may be provided in the form of a saltof a pharmaceutically-acceptable cation or esterified to form a C₁-C₁₆ester, or converted to an amide of formula NR₁R₂ wherein R₁ and R₂ areeach independently H or C₁-C₁₆ alkyl, or combined to form a heterocyclicring, such as a 5- or 6-membered ring. Amino groups of the protein,whether amino-terminal or side chain, may be in the form of apharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or may be modified to C₁-C₁₆ alkyl or dialkyl amino or furtherconverted to an amide.

[0146] Hydroxyl groups of the protein side chains may be converted toC₁-C₁₆ alkoxy or to a C₁-C₁₆ ester using well-recognized techniques.Phenyl and phenolic rings of the protein side chains may be substitutedwith one or more halogen atoms, such as fluorine, chlorine, bromine oriodine, or with C₁-C₁₆ alkyl, C₁-C₁₆ alkoxy, carboxylic acids and estersthereof, or amides of such carboxylic acids. Methylene groups of theprotein side chains can be extended to homologous C₂-C₄ alkylenes.Thiols can be protected with any one of a number of well-recognizedprotecting groups, such as acetamide groups. Those skilled in the artwill also recognize methods for introducing cyclic structures into theproteins of this invention to select and provide conformationalconstraints to the structure that result in enhanced stability.

[0147] Peptidomimetic and organomimetic embodiments are also within thescope of the present invention, whereby the three-dimensionalarrangement of the chemical constituents of such peptido- andorganomimetics mimic the three-dimensional arrangement of the proteinbackbone and component amino acid side chains in the apoptosis-modifyingfusion protein, resulting in such peptido- and organomimetics of theproteins of this invention having measurable or enhanced neutralizingability. For computer modeling applications, a pharmacophore is anidealized, three-dimensional definition of the structural requirementsfor biological activity. Peptido- and organomimetics can be designed tofit each pharmacophore with current computer modeling software (usingcomputer assisted drug design or CADD). See Walters, Computer-AssistedModeling of Drugs, in Klegerman & Groves (eds.), PharmaceuticalBiotechnology, Interpharm Press: Buffalo Grove, Ill., 165-174, 1993; andMunson (ed.) Principles of Pharmacology, Ch. 102, 1995, for descriptionsof techniques used in CADD. Also included within the scope of theinvention are mimetics prepared using such techniques that produceapoptosis-modifying fusion proteins.

IV. Activity of Fusion Proteins

[0148] Because the apoptosis modifying fusion proteins provided in thisinvention are at least bi-functional, having one domain required forcell targeting and another for modification of apoptosis in the targetcell, there are at least two activities for each fusion protein. Theseinclude the affinity of the fusion protein for a specific target cell,class of target cells, tissue type, etc., (the binding ability), and theability of the targeted fusion to effect apoptosis in the targeted cell(the apoptosis-modifying ability). Various techniques can be used tomeasure each of these activities.

[0149] A. Fusion Protein Affinity for Target Cells

[0150] Fusion protein affinity for the target cell, or to a specificcell surface protein, can be determined using various techniques knownin the art. One common method is a competitive binding assay (Greenfieldet al., Science 238:536-539, 1987). In a competitive binding assay,radiolabeled receptor binding protein, or a derivative or fragmentthereof, is exposed to the target native cell in the presence of one orvarying concentrations of cold fusion protein and other competitiveproteins being assayed. The amount of bound, labeled binding protein canbe measured through standard techniques to determine the relativecell-binding affinity of the fusion.

[0151] B. Apoptosis Inhibition or Enhancement

[0152] Several in vitro systems are used to study the process ofapoptosis. These include growth factor deprivation in culture, treatmentof cells with staurosporine (a non-specific protein kinase inhibitor),application of γ-radiation, and infection by viruses. Apoptosis asstimulated by any signal can be examined or measured in a variety ofways. Detection of morphological indicia of apoptosis (e.g., membraneblebbing, chromatin condensation and fragmentation, and formation ofapoptotic bodies) can provide qualitative information. More quantitativetechniques include TUNEL staining, measurement of DNA laddering,measurement of known caspase substrate degradation (e.g., PARP; Tayloret al., J. Neurochem. 68:1598-605, 1997) and counting dying cells, whichhave become susceptible to dye uptake. Many companies (e.g., Trevigen,Gaithersburg Md.; and R&D Systems, Minneapolis Minn.) also supply kitsuseful for the measurement of apoptosis by various methods; many ofthese kits can be used to measure the effect of disclosedapoptosis-modifying fusion proteins on apoptosis in a variety of celltypes.

[0153] By way of example, the following techniques can be used tomeasure the modification of apoptosis caused in a target cell after itis contacted with an apoptosis-modifying fusion protein of the presentinvention.

[0154] TUNEL staining: Terminal end-labeling of broken DNA fragmentswith labeled nucleotides; the reaction is catalyzed by terminalnucleotide transferase (TdT). Various kits are available for measurementof TUNEL staining, including the TdT in situ TUNEL-based Kit (R&DSystems, Minneapolis, Minn.).

[0155] Measurement of Caspase Activity: Another common system formeasuring the amount of apoptosis occurring in an in vitro cell systemis to measure the poly-ADP ribose Polymerase (PARP) cleavage aftertreatment of the cells with various stimulators of apoptosis. PARP is aknown substrate for a caspase (CPP-32) involved in the apoptotic kinasecascade. This technique can be carried out using essentially thefollowing protocol. HeLa cells are plated in growth media (e.g., EMEMcontaining 10% FBS at 2×10⁵ cells/ml) and treated with one or moreconcentrations of an apoptosis-modifying fusion protein according to thecurrent invention. The appropriate concentration for each fusion proteinwill depend on various factors, including the fusion protein inquestion, target cell, and apoptosis stimulator employed. Appropriateconcentrations may include, for instance, about 0.5 βM to about 3 μMfinal. It may be beneficial to treat the target cells multiple timeswith the fusion protein, usually after a period of incubation rangingfrom one to several hours. For instance, cells can be exposed to thefusion protein a second time about fifteen hours after the originaltreatment. Usually the same concentration(s) of fusion protein is usedin the second treatment.

[0156] Apoptosis is induced immediately the last treatment of the targetcells with apoptosis modifying fusion protein. The method of applicationof the apoptosis stimulus, amount applied, appropriate incubation timewith the inducer, etc., will be specific to the type of apoptosisinduction used (e.g., staurosporine, γ-radiation, virions, caspaseinhibitor, etc.). Such details are in general well known to those ofordinary skill in the art. After an appropriate incubation period, celllysates are prepared from the treated target cells, and aliquots loadedonto SDS-PAGE for analysis. The resultant gels can be examined using anyof various well-known techniques, for instance by performing a Westernanalysis immunoblotted with anti-PARP polyclonal antibody (BoehringerMannheim GmbH, Germany), developed with enhanced chemiluminescence.

[0157] Known inhibitors of apoptotic pathways, for instance caspaseinhibitors, can be used to compare the effectiveness ofapoptosis-modifying fusion proteins of this invention. Appropriateinhibitors include viral caspase inhibitors like crmA and baculovirusp35, and peptide-type caspase inhibitors including zVAD-fmk, YVAD- andDEVD-type inhibitors. See Rubin, British Med. Bulle., 53:617-631, 1997.

V. Incorporation of Apoptosis-Modifying Fusion Proteins intoPharmaceutical Compositions

[0158] Pharmaceutical compositions that comprise at least one apoptosismodifying fusion protein as described herein as an active ingredientwill normally be formulated with an appropriate solid or liquid carrier,depending upon the particular mode of administration chosen. Thepharmaceutically acceptable carriers and excipients useful in thisinvention are conventional. For instance, parenteral formulationsusually comprise injectable fluids that are pharmaceutically andphysiologically acceptable fluid vehicles such as water, physiologicalsaline, other balanced salt solutions, aqueous dextrose, glycerol or thelike. Excipients that can be included are, for instance, other proteins,such as human serum albumin or plasma preparations. If desired, thepharmaceutical composition to be administered may also contain minoramounts of non-toxic auxiliary substances, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like,for example sodium acetate or sorbitan monolaurate.

[0159] One or more other medicinal and pharmaceutical agents, forinstance chemotherapeutic, anti-inflammatory, anti-viral or antibioticagents, also may be included.

[0160] The dosage form of the pharmaceutical composition will bedetermined by the mode of administration chosen. For instance, inaddition to injectable fluids, topical and oral formulations can beemployed. Topical preparations can include eye drops, ointments, spraysand the like. Oral formulations may be liquid (e.g., syrups, solutionsor suspensions), or solid (e.g., powders, pills, tablets, or capsules).For solid compositions, conventional non-toxic solid carriers caninclude pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. Actual methods of preparing such dosage forms are known, orwill be apparent, to those skilled in the art.

[0161] The pharmaceutical compositions that comprise apoptosis modifyingfusion protein will preferably be formulated in unit dosage form,suitable for individual administration of precise dosages. One possibleunit dosage contains approximately 100 μg of protein. The amount ofactive compound administered will be dependent on the subject beingtreated, the severity of the affliction, and the manner ofadministration, and is best left to the judgment of the prescribingclinician. Within these bounds, the formulation to be administered willcontain a quantity of the active component(s) in an amount effective toachieve the desired effect in the subject being treated. Ideally, asufficient amount of the protein is administered to achieve tissue aconcentration at the site of action that is at least as great as invitro concentrations that have been shown to be effective.

VI. Clinical Use of Apoptosis-Modifying Fusion Proteins

[0162] The targeted apoptosis-regulating activity exhibited by thedisclosed fusion proteins makes these fusions useful for treatingneurodegenerative diseases, transient ischemic injuries, and unregulatedcell growth (as may for instance be found in tumors and various cancers

[0163] The apoptosis-modifying fusion proteins of this invention may beadministered to humans, or other animals on whose cells they areeffective, in various manners such as topically, orally, intravenously,intramuscularly, intraperitoneally, intranasally, intradermally,intrathecally, and subcutaneously. Administration of apoptosis-modifyingfusion protein composition is indicated for patients with aneurodegenerative disease, suffering from stroke episodes or transientischemic injury, or experiencing uncontrolled or unwanted cell growth,such as malignancies or neoplasms. More generally, treatment isappropriate for any condition in which it would be beneficial to alter(either inhibit or enhance) an apoptotic response of a subject's targetcells. The particular mode of administration and the dosage regimen willbe selected by the attending clinician, taking into account theparticulars of the case (e.g., the patient, the disease, and thedisease-state involved). By way of example, when apoptosis is beinggenerally inhibited over the short term, for instance after transientischemic neuronal injury, it may be advantageous to administerrelatively large doses of fusion protein repeatedly for a few days. Incontrast, if apoptosis is being enhanced in specific cell types, forinstance in hyper-proliferative cells, it may be of greater benefit toapply a relatively small dose of fusion protein repeatedly, e.g., daily,weekly, or monthly, over a much longer period of treatment.

[0164] In addition to their individual use, apoptosis-modifying fusionproteins as disclosed in the current invention may be combined withvarious therapeutic agents. For instance, an apoptosis-enhancing fusionprotein such as Bad-DTTR may be combined with or used in associationwith other chemotherapeutic or chemopreventive agents for providingtherapy against neoplasms or other hyper-proliferative cellular growthconditions. Various such anti-cancer agents are well known to those ofordinary skill in the art. Apoptosis-modifying fusion proteins accordingto this invention also can be supplied in the form of kits; theconstruction of kits appropriate for therapeutically active proteinsknown.

EXAMPLE 1 Construction of Functional Apoptosis-modifying Fusion Proteins

[0165] A. Bcl-x_(L)-DTR

[0166] The human Bcl-x_(L) gene from codon 1 through 233 (provided byDr. Craig Thompson) and the diphtheria toxin gene from codon 384 through535 (receptor binding domain, DTR), containing mutations in codons 508and 525, were amplified by PCR so that the DT mutation at codon 525 wasmutated to the wild-type by the PCR primer. The two PCR products,Bcl-x_(L)1-233 and DT384-535 (DTR), were digested with NdeI/NotI andNotI/XhoI restriction enzymes, respectively. Bcl-x_(L) was fused to the5′ end of the DTR gene with a linker (GCG TAT TCT GCG GCC GCG, SEQ IDNO: 5) to encode for Ala Tyr Ser Ala Ala Ala (SEQ ID NO: 6) between thetwo peptide domains. The two digested fragments were ligated into theprokaryotic expression vector pET 16b (Novagen, Inc., Madison, Wis.) cutwith NdeI and XhoI (FIG 1A). The codon 508 of DTR was mutated to thewild-type form (Phe→Ser) and the first three nucleotides (CAT) of NdeIwere deleted by double-stranded, site-directed mutagenesis. FIG 1A showsa schematic representation of the resultant apoptosis-modifying fusionprotein, Bcl-x_(L)-DTR.

[0167] As controls, human Bcl-x_(L) (codons 1-233) and DTR (codons384-535 of DT) genes were separately subcloned into pET16b vectorsthrough NdeI and XhoI sites. The histidine tag and Factor Xa digestionsite sequences from the expression vector were upstream of Bcl-x_(L),DTR and Bcl-x_(L)-DTR coding sequences. All three expression constructswere verified by sequencing.

[0168] For expression in eukaryotic cells, Bcl-x_(L)-DTR and Bcl-x_(L)gene constructs were inserted in the eukaryotic vector pcDNA3(Invitrogen, Carlsbad, Calif.) and the constructs verified bysequencing.

[0169] B. Bad-DTTR

[0170] The full-length mouse Bad gene with two Ser→Ala mutations atcodons 112 and 136 (Schendel et al., Proc. Natl. Acad. Sci. USA94:5113-5118, 1997), and the diphtheria toxin gene from codons 194through 535 (translocation and receptor-binding domains, DTTR, withoutthe catalytic domain) were amplified by PCR. The two PCR products, Badand DT194-535 (DTTR), were used as templates to directly fuse the Badgene to the 5′ end of DTTR gene by a second round of PCR. The Bad-DTTRgene fragment was digested with NdeI and XhoI and ligated into theprokaryotic expression vector pET 16b (Novagen, Inc., Madison, Wisc.)digested with NdeI and XhoI. The histidine tag and Factor Xa digestionsite sequences from the expression vector were upstream of the Bad-DTTRcoding sequence. The expression construct was verified by sequencing.

EXAMPLE 2 Expression and Purification of Functional Apoptosis-modifyingFusion Proteins

[0171] A. Prokaryotic Expression

[0172] To produce proteins for extracellular addition to cells, theBcl-x_(L) gene, the DTR domain gene and the Bcl-x_(L)-DTR fusion genewere cloned into pET16b. E. coli BL21(DE3) strain was used to expressBcl-x_(L)-DTR, Bad-DTTR, Bcl-x_(L) and DTR, with addition of 1 mM IPTGwhen the OD260 reached 0.5-0.7. After two hours incubation and lysis byFrench press the inclusion bodies were collected and dissolved in 6Mguanidine-HCl.

[0173] B. Eukaryotic Expression

[0174] Transfection of HeLa cells with the fusion constructs wasperformed as reported previously (Wolter et al., J Cell Biol139:1281-1292, 1997). HeLa cells were harvested and lysed in 1 ml buffercontaining 100 μg/ml leupeptin 20 hours after transfection, centrifugedto remove cell debris, and 15 μl aliquots of the supernatant loaded onto10-20% SDS-PAGE. The plasmid encoded proteins were visualized byimmunoblotting with anti-Bcl-x_(L) monoclonal antibody (2H12, Trevigen,Gaithersburg, Md.) and developed using enhanced chemiluminescence(Amersham Inc., Arlington Heights, Ill.). Results are shown in FIG. 1B.

[0175] C. Purification

[0176] Histidine tag binding resin (Novagen, Inc., Madison, Wis.) wasused to purify Bcl-x_(L)-DTR, Bad-DTTR, Bcl-x_(L), and DTR. Proteinswere refolded by dialysis against, or dilution into, 100 mM Tris-Acetate(pH 8.0)/0.5 M arginine, concentrated with PEG15,000-20,000 and dialyzedagainst PBS. This yielded protein purified to greater than 90%homogeneity. The four proteins were subjected to 10-20% SDS-PAGE,visualized by immunoblotting with either anti-Bcl-x_(L) monoclonal(2H12) or horse anti-DT polyclonal antibodies (Centers for DiseaseControl, Atlanta, Ga.) and developed as above. They were of the expectedmolecular weight on SDS PAGE and of the expected immunoreactivity toantibodies against Bcl-x_(L) or DT on Western blots.

EXAMPLE 3 Assays for Measuring Fusion Protein Binding to, andTranslocation Into, Target Cells

[0177] A. Competitive Binding Assay

[0178] Protein binding to the diphtheria toxin receptor was performed aspreviously reported (Greenfield et al., Science 238:536-539, 1987) withthe following modifications. DT was radiolabeled with I¹²⁵ usingiodobeads (Pierce Chem. Co., Rockford, Ill.) as described by themanufacturer. Cos-7 cells, grown to confluency in 12 well costar plates,were analyzed for receptor binding and competition by incubation forthree hours on ice. Results are reported in FIG. 2. Cold competitorproteins, native DT (Δ), Bcl-x_(L)-DTR (▴), Bcl-x_(L) (∘), and DTR (),were used to displace I¹²⁵ labeled DT tracer.

[0179] Native DT and Bcl-x_(L)-DTR compete for DT receptor binding inthe nanomolar concentration range. DT and the Bcl-x_(L)-DTR fusionprotein competed for I¹²⁵-DT binding to its receptor to a similar extentalthough the affinity of the fusion was three times lower than that ofnative DT (FIG. 2). Neither the Bcl-x_(L) domain alone nor the DTRdomain alone was able to compete for DT receptor binding. The morecomplete protein (Bcl-x_(L)-DTR), where Bcl-x_(L) is substituted for theDT translocation domain, folded such that DT receptor binding activitywas retained whereas the isolated binding domain (DTR) did not. Additionof the DT A chain domain to the N-terminus of Bcl-x_(L)-DTR furtherincreased the affinity of the chimera to the DT receptor.

[0180] B. Assays for Effective Transport of the Fusion Protein Into theTarget Cell

[0181] Diphtheria toxin is endocytosed by cells and reaches low pHintracellular compartments. The low pH triggers a conformational changein the translocation domain, which allows this domain to insert intomembranes and form channels. The toxicity of DT is blocked bylysosomotropic agents such as chloroquine, which increase the pH ofintracellular compartments. Chloroquine at a concentration that blocksdiphtheria toxin toxicity (10 μM) did not block the activity ofBcl-x_(L)-DTR to inhibit poliovirus-induced cell death. Thus, themechanism of membrane interaction of Bcl-x_(L)-DTR differs to someextent from that of DT. However, brefeldin A, an inhibitor of vesicletraffic between the ER and the Golgi apparatus (Lippincott-Schwartz etal., Cell 67:601-616, 1991; Hunziker et al, Cell 67:617-627, 1991), doesblock the anti-apoptosis activity of Bcl-x_(L)-DTR (Table 3). Theseresults indicate that Bcl-x_(L)-DTR must be endocytosed and suggest thatBcl-x_(L)-DTR must reach the Golgi apparatus or the ER to prevent celldeath. The subcellular location from which native Bcl-2 family membersregulate apoptosis is currently under scrutiny (Hunziker et al., Cell67:617-627, 1991). Several intracellular membrane locations, includingthe ER, appear able to mediate Bcl-2 family regulation of cell death(Krajewski et al., Cancer Res. 53:4701-4714, 1993). Bcl-x_(L)-DTR mayreach the ER to translocate into the cell cytosol or perhapsBcl-x_(L)-DTR, when bound closely to a membrane, can insert into thatmembrane and inhibit apoptosis in the membrane-intercalated form.

EXAMPLE 4

[0182] Measurement of Bcl-x_(L)-DTR Apoptosis-inhibiting Activity

[0183] A. Apoptosis Inhibition After Transient Cell Transfection

[0184] To demonstrate that Bcl-x_(L)-DTR is effective at inhibitingapoptosis when expressed from within the target cell, this construct andthe control construct containing Bcl-x_(L) were transiently transfectedinto HeLa cells. Assay of apoptosis inhibition after transienttransfection was performed as reported previously (Wolter et al., J.Cell Biol. 139:1281-1292, 1997). The Bcl-x_(L)-DTR fusion gene blockedapoptosis after transient transfection into HeLa cells (FIG. 1C) to anextent similar to that of the Bcl-x_(L) gene after C-terminal tailtruncation (Wolter et al., J Cell Biol 139:1281-1292, 1997).

[0185] B. Inhibition of STS-induced Apoptosis by Extracellular Treatmentwith Bcl-x_(L)-DTR

[0186] Hoechst dye no. 33342 staining: The effectiveness ofextracellular delivery of Bcl-x_(L) or the Bcl-x_(L)-DTR fusion proteinfor inhibiting the rate of cell death by apoptosis was examined asfollows. Cos-7 cells at 3×10⁴ cells/cm² in 100 μl DMEM with 10% FBS wereincubated with 0.1 μM STS (∘) 0.1 μM STS plus 4.8 μM Bcl-x_(L)-DTRprotein added to the medium (Δ) or 20 μl of PBS (□). Apoptotic cellswere quantified by staining with Hoechst dye no. 33342. Results in FIG.3A are presented as the average number of cells per field (magnification160×). For each point, at least 5 fields were counted in each of atleast 3 wells. Bcl-x_(L)-DTR dramatically decreased the rate ofapoptosis in Cos-7 cells. Six different preparations of Bcl-x_(L)-DTRwere found to have activity and the apoptosis prevention activity wasstable for at least 5 months when Bcl-x_(L)-DTR was stored at 4° C.Addition of Bcl-x_(L)-DTR minutes before the addition of STS blockedmore than 70% of Cos-7 cell death after 6 hours and more than 50% ofcell death after 12 hours of STS exposure (FIG. 3A).

[0187] Jurkat, HeLa and U251 cells were also protected from STS-inducedapoptosis by Bcl-x_(L)-DTR (Table 2). Bcl-x_(L) protein added to Cos-7cells, however, did not alter the extent of cell death induced by STS. Anontoxic DT mutant able to bind the DT receptor, CRM197, also had noeffect on apoptosis induced by STS. To further test the role of DTreceptor binding in apoptosis inhibition, cells expressing DT receptorswere compared with cells lacking DT receptors. Mouse and rat cells arethousands of times less sensitive to DT than human or monkey cell linesdue to a lack of the DT receptor (Pappenheimer The Harvey Lectures76:45-73, 1982). Comparing human, monkey, mouse and rat cell linesrevealed that those cells lacking the DT receptor, WEHI-7.1 and 9L, wereinsensitive to apoptosis protection by Bcl-x_(L)-DTR (Table 2). Thesensitivity of the six cell lines to DT toxicity, thought to reflect DTreceptor levels, correlated with sensitivity to apoptosis prevention byBcl-x_(L)-DTR (Table 2).

[0188] The magnitude of apoptosis inhibition by extracellularBcl-x_(L)-DTR (FIG. 3A, Table 2) was similar to that found bytransfection of the fusion gene into cells (FIG. 1C). Although fusion tothe C-terminus of Bcl-x_(L) inhibited bioactivity relative to nativeBcl-x_(L) after transfection (FIG. 1C), a very substantial prevention ofcell death was obtained at both the gene level and the protein level(FIG. 3A). Thus the delivery of Bcl-x_(L)-DTR is efficient and apoptosiscan be prevented by delivery of Bcl-x_(L) from the outside of cells.

[0189] Measurement of caspase activity: To confirm the results of celldeath measurements by Hoechst staining and trypan blue dye exclusion, weexamined caspase-induced cleavage of poly-ADP ribose polymerase (PARP).HeLa cells were plated in EMEM containing 10% FBS at 2×10⁵ cells/ml andtreated with two different preparations of Bcl-x_(L)-DTR at 1.48 μM or 1μM. Fifteen hours later, cells were treated again with Bcl-x_(L)-DTR at1.48 μM or 1 μM. Immediately after the second treatment, 0.8 μM STS wasadded. Three hours later, cell lysates were made and aliquots wereloaded onto SDS-PAGE, immunoblotted with anti-PARP polyclonal antibody(Boehringer Mannheim GmbH, Germany) and developed with enhancedchemiluminescence. Lane a contains control HeLa cells not incubated withSTS (uninduced cells); Lane b, HeLa cells treated with STS plus 1 μMBcl-x_(L)-DTR protein; Lane c, HeLa cells treated with STS plus 1.48 μMBcl-x_(L)-DTR protein; and Lane d, HeLa cells treated with STS and nofusion protein. HeLa cells incubated with Bcl-x_(L)-DTR showedsignificantly less cleavage of PARP after apoptosis induction with STS(FIG. 3B).

[0190] C. Inhibition of γ-radiation-induced Apoptosis by ExtracellularTreatment with Bcl-x_(L)-DTR

[0191] Radiation is a potent inducer of apoptosis in many hematopoeticcell types. The ability of Bcl-x_(L)-DTR to prevent radiation-inducedapoptosis was examined in the human T cell line, Jurkat. When added tothe media (serum-free RPMI-1640 medium with insulin and transferrin) ofJurkat cells plated at 10⁵ cells/ml a few minutes prior to induction ofapoptosis by 10 gray γ-radiation, Bcl-x_(L)-DTR (4.63 μM) blocked almosthalf of the ensuing cell death (FIG. 4A). Apoptotic cells were countedusing Hoechst dye no. 33342. Control cells were not irradiated and nottreated with Bcl-x_(L)-DTR.

[0192] In a clonogenic assay measuring long term survival, Jurkat cellsshowed more than a 3-fold greater survival when Bcl-x_(L)-DTR was addedto the media immediately prior to 5 gray γ-radiation.

[0193] D. Inhibition of Anti-Fas-induced Apoptosis by ExtracellularTreatment with Bcl-x_(L)-DTR

[0194] Jurkat cells are also sensitive to apoptosis induced by antibodybinding to the Fas/APO-1/CD95 receptor. The Fas pathway of apoptosis isone of the few pathways shown to be less sensitive or insensitive toapoptosis protection by Bcl-2 and Bcl-x_(L) (Boise & Thompson Proc.Natl. Acad. Sci. USA 94:3759-3764, 1997; Memon et al., J Immunol.155:4644-4652, 1995) and contrasts with radiation-induced apoptosis inthis regard. Jurkat cells were plated at 10⁵ cells/ml in serum-freeRPMI-1640 medium with insulin and transferrin, and treated with 100ng/ml anti-Fas antibody (CH 11, Upstate Biotechnology, Lake Placid,N.Y.) minutes after addition of Bcl-x_(L)-DTR to a concentration 4.68μM. Control cells were treated with PBS and no anti-Fas antibody. Fasantigen-induced apoptosis (measured by counting dying cells usingHoechst dye no. 33342) showed very little inhibition by Bcl-x_(L)-DTR,although there was a statistically significant decrease in apoptosisbetween 2 and 4 hours in some experiments (FIG. 4B). The degree ofprotection of different apoptosis pathways by extracellularBcl-x_(L)-DTR corresponded with that seen by transfection with theBcl-x_(L) gene.

[0195] E. Inhibition of Poliovirus-induced Apoptosis by ExtracellularTreatment with Bcl-x_(L)-DTR

[0196] Viruses induce a powerful apoptosis response in certain cells andprevention of this apoptosis may have therapeutic utility (Hardwick,Adv. Pharm. 41:295-336, 1997). Poliovirus-induced apoptosis of HeLacells was also examined for sensitivity to extracellular Bcl-x_(L)-DTR,a system where inhibition of cell death by transfection with theBcl-x_(L) gene has been demonstrated (Castelli et al., J Exp. Med.186:967-972, 1997). Adding Bcl-x_(L)-DTR 30 minutes after infection ofcells with low titers (MOI of 1 pfu/cell) of poliovirus (FIG. 5) or withmoderately high titers (MOI of 20 pfu/cell) of poliovirus prevented morethan half of the cell death for up to 24 hours. Addition ofextracellular Bcl-x_(L) or the DTR domain proteins alone had no affecton poliovirus-induced apoptosis.

[0197] F. Competition of Apoptosis Inhibition

[0198] Caspase inhibitors block many pathways of apoptosis and are beingexplored for pharmacologic potential to inhibit cell death (Chen et al.,Nature 385:434-439, 1997). zVAD-fmk and Boc-D-fink are powerful, broadspecificity caspase inhibitors that block many apoptosis pathways(Henkart, Immunity 4:195-201, 1996). Apoptosis inhibition activity ofzVAD-fmk and Boc-D-fink was compared with that of Bcl-x_(L)-DTR. HeLacells were plated at a density of 1×10⁵ cells/well in EMEM containing10% FBS and antibiotics, infected with poliovirus at an MOI of 1pfu/cell as reported previously (Castelli et al., J Exp Med 186:967-972,1997) and immediately treated with negative control peptide zFA-fmk at20 μM, Bcl-x_(L)-DTR at 0.48 μM, or peptides zVAD-fmk or Boc-D-fmk at 20μM. Cell viability was assessed by trypan blue dye exclusion 24 hoursfollowing addition of virus. zFA-fmk, zVAD-fmk and Boc-D-fmk were fromEnzyme Systems Products, Dublin, Calif.

[0199] Bcl-x_(L)-DTR at 0.48 μM blocked cell death to a greater extentthan either zVAD-fmk or Boc-D-fmk at 20 μM (FIG. 5). Bcl-x_(L)-DTRshowed a strong inhibition of a potent and pathologically importantapoptosis pathway. Interestingly, Bcl-x_(L) appears to act at an earlystep in the cell death pathway when intervention can permit long termviability of cells, whereas caspase inhibitors appear to work relativelymore downstream in the apoptosis pathway (Chinnaiyan et al., J. BiolChem 271:4573-4576, 1996; Xiang et al., Proc. Natl. Acad. Sci. USA93:14559-14563, 1996; Miller et al., J Cell Biol 139:205-217, 1997).

EXAMPLE 5 Measurement of Bad-DTTR Apoptosis-enhancing Activity

[0200] A. Stimulation of Apoptosis by Extracellular Treatment withBad-DTTR

[0201] To determine the effectiveness of the fusion protein Bad-DTTR attriggering apoptosis, cell survival after exposure to Bad-DTTR wasexamined. U251 MG cells at 3×10⁴ cells/cm² in 100 μl DMEM with 10% FBSwere incubated with 0.65 μM Bad-DTTR protein added to the medium or 20μl of PBS. Total and apoptotic cells were quantified by staining withHoechst dye no. 33342. Results are presented in FIG. 6 as the averagenumber of cells per field (magnification 160×). Bad-DTTR decreases cellviability 12 hours after treatment.

[0202] B. Enhancement of STS-triggered Apoptosis by ExtracellularTreatment with Bad-DTTR

[0203] To examine the ability of Bad-DTTR to enhance apoptosis triggeredby STS, cell survival was determined after exposure to variousconcentrations of STS, in combination with various combinations ofBad-DTTR. U251 MG cells at 3×10⁴ cells/cm² in 100 μl DMEM with 10% FBSwere treated with PBS, 0.1μM STS, 0.65 μM Bad-DTTR, 0.065 μM Bad-DTTR,0.1 μM STS plus 0.65 μM Bad-DTTR and 0.1 μM STS plus 0.065 μM Bad-DTTR.Apoptotic death cells were quantified at different times by stainingwith Hoechst dye no. 33342. Results are presented as the average numberof cells per field (magnification 160×). Apoptosis is most enhanced whencells are treated with 0.1 μM STS plus 0.65 μM Bad-DTTR, and cells beginto die about 12 hours after treatment.

[0204] U251 MG cells at 3×10⁴ cells/cm² in 100 μl DMEM with 10% FBS weretreated with PBS, 1 μM STS, 0.65 μM Bad-DTTR, 0.065 μM Bad-DTTR, 1 μMSTS plus 0.65 μM Bad-DTTR and 1 μM STS plus 0.065 μM Bad-DTTR. Apoptoticcells were quantified and presented as above. The combination of 1 μMSTS and Bad-DTTR at various concentrations causes an earlier onset ofapoptosis in U251 MG cells.

EXAMPLE 6 LF_(n)-Bcl-x_(L) Inhibits Neuron, Macrophage, and LymphocyteApoptosis

[0205] Anthrax toxin includes three components: lethal factor (LF),edema factor (EF) and protective antigen (PA) (Leppla, Anthrax toxin. InHandbook of Natural Toxins, Moss et al., Eds., Dekker, N.Y., Vol. 8, pp.543-572, 1995). PA binds simultaneously to LF and to a cell surfacereceptor existing on the cells of almost all species including rodents(Leppla, 1995; Friedlander, J. Biol. Chem. 261:7123-7126, 1986), andtransports LF into cells where LF causes toxic effects. PA alone,however, is not toxic. It has been found that the first 255 residues(LF_(n)) of LF, which constitute the PA-binding domain and are not toxicto cells, are sufficient for delivery of heterologous peptides to thecytosol. Cytotoxins have been fused to LF_(n) (Leppla, 1995; Arora etal., J Biol. Chem. 269:26165-26171, 1994; Milne et al., Mol. Microbiol.15: 661-666, 1995). Administration of a fusion protein containing LF_(n)and the gp 120 envelope glycoprotein of HIV-1 along with PA toantigen-presenting cells sensitized them to cytolysis by cytotoxicT-lymphocytes (CTL) specific to gp120 (Goletz et al., Proc Natl Acad SciUSA 94:12059-12064, 1997). In vivo, LF_(n)-fused to CTL epitopesinjected along with PA has been shown to stimulate a CTL responseagainst the antigens in mice (Ballard et al., Proc. Natl. Acad. Sci. USA93: 12531-12534, 1996; Ballard et al., Infect. Immun. 66:615-619, 1998;Ballard et al., Infect. Immun. 66:4696-4699, 1998; Doling et al.,Infect. Immun. 67: 3290-3296, 1999).

[0206] To inhibit neuron apoptosis, another protein delivery system wasengineered by fusing a nontoxic domain of anthrax toxin to Bcl-x_(L), tocreate the LF_(n)-Bcl-x_(L) chimeric fusion protein. Macrophage andlymphocyte death in culture, and neuron death in vivo in a retinalganglion cell model of apoptosis induced by axotomy, can be prevented byapplication of this fusion protein.

[0207] A. Construction of LF_(n)-Bcl-x_(L) in a Prokaryotic ExpressionPlasmid

[0208] The coding sequence for lethal factor (LF) from codons 34 to 288(LF_(n)) (Bragg et al., Gene 81:45-54, 1989), which is theamino-terminal domain (residues 1-255) of mature LF (Leppla, 1995), wasamplified using PCR with the template of pET15b/LF_(n) (Milne et al.,Mol. Microbiol. 15: 661-666, 1995). The gene of human Bcl-x_(L) fromCodons 1 to 209 (Bcl-x_(L)(1-209)) (Boise et al., Cell 74: 597-608,1993) was amplified by PCR. Then the LF_(n) encoding sequence was fusedto the 5′ end of Bcl-x_(L)(1-209) encoding sequence by a second round ofPCR. A stop codon was introduced immediately after Codon 209 ofBcl-x_(L). The fused DNA fragment, LF_(n)-Bcl-x_(L), was cut with NdeIand Xho I, and inserted into prokaryotic expression vector pET15b cutwith Nde I and Xho I (FIG. 8). A histidine tag and thrombin cleavagesite were linked to the N-terminal of LF_(n)-Bcl-x_(L). Similarly, theBcl-x_(L) gene from codons 1 to 209 was also genetically inserted intopET15b at the sites of Nde I and Xho I. All the constructs were verifiedby DNA sequencing.

[0209] B. Construction of Ekaryotic Expression Plasmids, Transfection,Western Blotting and Biologic Activity Assay

[0210] The sequences encoding LF_(n)-Bcl-x_(L), Bcl-x_(L) from codons 1to 209, and full-length Bcl-x_(L), were separately engineered intoeukaryotic expression vector pcDNA3.1+ and verified by DNA sequencing.Cos-7 cells were co-transfected with plasmid EGFP-C3 and one of thethree plasmids as reported (Keith et al., J Cell Biol 139: 1281-1292,1997). The cells were treated with 0.1 μM staurosporine (STS) 12 hourslater. The dead and living cells were counted with Hoechst 33342 atdifferent times after STS treatment (Liu et al., Proc Natl Acad Sci USA96: 9563-9567, 1999; Keith et al., J Cell Biol 139: 1281-1292, 1997).The cells were harvested and lysed 20 hours after transfection, andaliquots were loaded onto SDS/10-20% PAGE gels. The plasmid-encodedproteins were visualized by immunoblotting with anti-Bcl-x_(L) mAb(Trevigen, Gaithersburg, Md.) and developed by using enhancedchemiluminescence (Amersham Pharmacia).

[0211] C. Protein Expression, Purification, SDS-PAGE and WesternBlotting

[0212] The proteins LF_(n), LF_(n)-Bcl-x_(L) and Bcl-x_(L) from codons 1to 209 were individually expressed in E. coli BL21(DE3) (Novagen, Inc.)and purified with a His.Tag binding purification kit (Novagen, Inc.).The transformed BL21(DE3) was cultured at 37° C. in LB medium until theOD600 reached 0.5-0.8, and treated with 1 mM IPTG, and then cultured for3 more hours. The cells was pelleted, suspended in 1×His.Tag bindingbuffer with 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM aprotininand 1 mM leupeptin, and disrupted with French Press. The cytosol wasseparated from cell debris and undisrupted cells by centrifugation at20,000×g for 30 minutes and loaded on the His.Tag binding column. Theeluted proteins were dialyzed against 1×PBS and sterilized with 0.22-umfilter. Protective antigen (PA) was purified as reported (Milne et al.,Mol. Microbiol. 15: 661-666, 1995). The proteins were run on SDS-PAGEgels, and stained with Coomassie Blue or visualized by immunoblottingwith anti-Bcl-x_(L) antibody, and developed as above.

[0213] D. J744 Macrophage-like Cell Culture, Treatment and ApoptosisAssay

[0214] J744 macrophage-like cells at 10⁵/ml were placed in 96-wellplates (100 μl per well), and cultured overnight in RPMI 1640 with 10%FCS. The cells were treated with PBS, 0.1 μM staurosporine alone or 0.1μM staurosporine along with the different combinations of the proteinsLF_(n)-Bcl-x_(L) (28 μg/ml), PA (33 μg/ml), LF_(n) (28 μg/ml) andBcl-x_(L) (28 μg/ml). The apoptotic and living cells were counted withHoechst dye no. 33342 as reported (Liu et al., Proc Natl Acad Sci USA96: 9563-9567, 1999).

[0215] E. Optic Nerve Section and Intra-ocular Protein Injection

[0216] The P0 pups of Fisher 344 rat strain were used for the presentstudy. P0 is defined as the day of birth. The intracranial lesion ofunilateral optic nerve was performed as reported (Rabachi et al., JNeurosci. 14: 5292-301, 1994). Briefly, a P0 pup was anesthetized byhypothermia. Under a dissecting microscope, an incision over the righteye was cut and a piece of bone flipped up. The right optic nerve wassectioned after suctioning the overlying cerebral cortex. The sectionsite of optic nerve is about 3 mm away from the eyeball. A piece ofgelfoam was put in the hole, and the flipped bone replaced, and theincision repaired with SUPERGLUE™. Immediately after the operation,seven, ten and four mice were respectively treated with administrationof PBS, LF_(n)-Bcl-x_(L) (0.65 μg) plus PA (0.35 μg) and PA (0.35 μg) ina volume of 350 nanoliters (nl) per eye through ora serrata into theposterior chamber of the right eyes by using a micro-injector with apulled micropipette. The pups were warmed up with a light lamp until therecovery, and then sent back to the mother. Four pups from the samelitters, which were not operated and not treated, were used for normalcontrol.

[0217] F. Histology

[0218] About 24 hours after sectioning of the optic nerve, the righteyes were removed under deep anesthesia with sodium pentobarbital, fixedin 4% paraformaldehyde for approximately 30 hours, embedded in paraffinand cut at 6 μm. The eyes taken from the normal pups in the same litterswere processed in the same way to serve as controls. The sections wererehydrated, stained with 0.2% cresyl violet, dehydrated, and mountedwith DPX mountant. The number of pyknotic cells and the number of livingcells were counted by the use of 40× objective in the entire retinalganglion cell layer of three sections per retina. The pyknotic cellswere identified as reported (Rabachi et al., J. Neurosci. 14: 5292-301,1994). The values were presented as the percentage of pyknotic cellsversus total cells per retina (FIG. 12).

[0219] G. Results

[0220] The PA protein from the Anthrax bacillus binds cell receptors andcan mediate the delivery of the anthrax LF protein to the cell cytosolwhere LF effects toxicity to cells. The N-terminal domain of LF binds toPA. When exogeneous peptides are fused to the N-terminal domain of LF(LF_(n)), they can be delivered to the cell cytosol by PA. Deletion ofthe C-terminal region of LF prevents toxicity to cells. To deliverBcl-x_(L) to cells, the N-terminal 255 amino acids of LF were fused toBcl-x_(L) without including the C-terminal 24 hydrophobic amino acids ofBcl-x_(L), as shown schematically in FIG. 8. The nucleotide and aminoacid sequences of the fusion protein, LF_(n)-Bcl-x_(L), are shown in SEQID NOs: 7 and 8. The fusion protein was expressed in E. coli andpurified to near homogeneity.

[0221] The bioactivity of the LF_(n)-Bcl-x_(L) was explored in J774cells in tissue culture. LF_(n)-Bcl-x_(L), at 28 micrograms per ml plusPA at 33 micrograms per ml was added to the media of cells at the timeof apoptosis induction with 0.1 μM staurosporine (STS). Cells treatedwith staurosporine alone died by apoptosis over the following 36 hoursas shown in FIG. 9. When the cells were treated with LF_(n)-Bcl-x_(L)plus PA, most of the cell death was inhibited.

[0222] Controls were performed to explore the requirements for apoptosisinhibition. FIG. 10 shows data demonstrating that J774 cells treatedwith LF_(n) alone, Bcl-x_(L) alone, LF_(n)-Bcl-x_(L) without PA, and PAwithout LF_(n)-Bcl-x_(L) were not protected from apoptosis induced bystaurosporine, whereas LF_(n)-Bcl-x_(L) plus PA prevented more than halfof the cell death. Jurkat cells were also protected from apoptosis byLF_(n)-Bcl-x_(L) plus PA (FIG. 11).

[0223] This new strategy to block cell death was explored in an in vivomodel of neuron apoptosis. Retinal ganglion cells were axotomized andimmediately afterwards a mixture containing 0.35 μg of PA and 0.65 μg ofLF_(n)-Bcl-x_(L) was injected into the eye. Control mice were either notaxotomized, axotomized and injected with PBS, or axotomized and injectedwith PA alone. Mice were sacrificed 24 hours later, and the eyesexamined histologically. An increase in pyknotic cells, i.e., apoptoticcells (Rabachi et al., J Neurosci. 14: 5292-301, 1994), occurs in theganglion layer 24 hours after axotomy. However, when eyes are injectedwith LF_(n)-Bcl-x_(L) and PA, much of the cell death is inhibited. PAalone did not prevent cell death. To quantitate the extent of celldeath, the number of living and pyknotic cells in three entire ganglionlayers in one eye from each of 4-10 mice was counted. The quantifiedresults are shown in FIG. 12. LF_(n)-Bcl-x_(L) inhibited more than halfof the cell death due to neuron axotomy in vivo.

[0224] In view of the many possible embodiments to which the principlesof our invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention,and should not be taken as limitations on its scope. Rather, the scopeof the invention is defined by the following claims. We therefore claimas our invention all that comes within the scope and spirit of theseclaims. TABLE 2 Inhibition of Apoptosis by Bcl-x_(L)-DTR Concentrationof Time of STS Apoptosis Apoptosis Bcl-x_(L)-DTR Treatment Prevention DTCell line inducer (μM) (Hrs) (%*) IO₅₀ (M) Cos-7 0.1 μM STS 4.8 12 58.410⁻¹²-10⁻¹¹ (monkey kidney) U251 0.1 μM STS 4.68 16 57.5 10⁻¹²-10⁻¹¹(human glioma) HeLa 0.2 μM STS 2.17 10 32.4 10⁻¹²-10⁻¹¹ (human cervicalCa) Jurkat 0.1 μM STS 4.68 12 21.2   10⁻⁹ (human T leukemia) 9L 0.1 μMSTS 4.68 12 −5.4 >10⁻⁹ (rat gliosarcoma) WEH7.1 0.1 μM STS 4.68 12 0.5>10⁻⁷ (mouse T lymphoma) # except for the non-adherent Jurkat andWEHI7.1 cells which were counted by trypan blue dye exclusion and %apoptosis prevention calculated as (number of living cells withSTS)/(number of living cells without STS and Bcl-x_(L)-DTR).

[0225] TABLE 3 Brefeldin A prevents Bcl-x_(L)-DTR blockade of apoptosis0.1 μM STS + PBS 0.1 μM STS 2.24 μM Bcl-X_(L)-DTR Bcl-x_(L)-DTR Celldeath (%) 1 24 11 56% protection 0.1 μM STS + 2 μM 0.1 μM STS + 2 μMbrefeldin A + Bcl-X_(L)-DTR + brefeldin A 2 μM brefeldin A 2.24 μMBcl-x_(L)-DTR brefeldin A Cell death (%) 2 35 32 9% protection #withoutSTS and Bcl-x_(L)-DTR)/(number of apoptotic cells with STS-number ofapoptotic cells without STS and Bcl-x_(L)-DTR).

[0226]

1 8 1 1236 DNA Artificial Sequence Description of Artificial Sequencegenetic fusion 1 atg ggc cat cat cat cat cat cat cat cat cat cac agc agcggc cat 48 Met Gly His His His His His His His His His His Ser Ser GlyHis 1 5 10 15 atc gaa ggt cgt atg tct cag agc aac cgg gag ctg gtg gttgac ttt 96 Ile Glu Gly Arg Met Ser Gln Ser Asn Arg Glu Leu Val Val AspPhe 20 25 30 ctc tcc tac aag ctt tcc cag aaa gga tac agc tgg agt cag tttagt 144 Leu Ser Tyr Lys Leu Ser Gln Lys Gly Tyr Ser Trp Ser Gln Phe Ser35 40 45 gat gtg gaa gag aac agg act gag gcc cca gaa ggg act gaa tcg gag192 Asp Val Glu Glu Asn Arg Thr Glu Ala Pro Glu Gly Thr Glu Ser Glu 5055 60 atg gag acc ccc agt gcc atc aat ggc aac cca tcc tgg cac ctg gca240 Met Glu Thr Pro Ser Ala Ile Asn Gly Asn Pro Ser Trp His Leu Ala 6570 75 80 gac agc ccc gcg gtg aat gga gcc act gcg cac agc agc agt ttg gat288 Asp Ser Pro Ala Val Asn Gly Ala Thr Ala His Ser Ser Ser Leu Asp 8590 95 gcc cgg gag gtg atc ccc atg gca gca gta aag caa gcg ctg agg gag336 Ala Arg Glu Val Ile Pro Met Ala Ala Val Lys Gln Ala Leu Arg Glu 100105 110 gca ggc gac gag ttt gaa ctg cgg tac cgg cgg gca ttc agt gac ctg384 Ala Gly Asp Glu Phe Glu Leu Arg Tyr Arg Arg Ala Phe Ser Asp Leu 115120 125 aca tcc cag ctc cac atc acc cca ggg aca gca tat cag agc ttt gaa432 Thr Ser Gln Leu His Ile Thr Pro Gly Thr Ala Tyr Gln Ser Phe Glu 130135 140 cag gta gtg aat gaa ctc ttc cgg gat ggg gta aac tgg ggt cgc att480 Gln Val Val Asn Glu Leu Phe Arg Asp Gly Val Asn Trp Gly Arg Ile 145150 155 160 gtg gcc ttt ttc tcc ttc ggc ggg gca ctg tgc gtg gaa agc gtagac 528 Val Ala Phe Phe Ser Phe Gly Gly Ala Leu Cys Val Glu Ser Val Asp165 170 175 aag gag atg cag gta ttg gtg agt cgg atc gca gct tgg atg gccact 576 Lys Glu Met Gln Val Leu Val Ser Arg Ile Ala Ala Trp Met Ala Thr180 185 190 tac ctg aat gac cac cta gag cct tgg atc cag gag aac ggc ggctgg 624 Tyr Leu Asn Asp His Leu Glu Pro Trp Ile Gln Glu Asn Gly Gly Trp195 200 205 gat act ttt gtg gaa ctc tat ggg aac aat gca gca gcc gag agccga 672 Asp Thr Phe Val Glu Leu Tyr Gly Asn Asn Ala Ala Ala Glu Ser Arg210 215 220 aag ggc cag gaa cgc ttc aac cgc tgg ttc ctg acg ggc atg actgtg 720 Lys Gly Gln Glu Arg Phe Asn Arg Trp Phe Leu Thr Gly Met Thr Val225 230 235 240 gcc ggc gtg gtt ctg ctg ggc tca ctc ttc agt cgg aaa gcgtat tct 768 Ala Gly Val Val Leu Leu Gly Ser Leu Phe Ser Arg Lys Ala TyrSer 245 250 255 gcg gcc gcg cat aaa acg caa cca ttt ctt cat gac ggg tatgct gtc 816 Ala Ala Ala His Lys Thr Gln Pro Phe Leu His Asp Gly Tyr AlaVal 260 265 270 agt tgg aac act gtt gaa gat tcg ata atc cga act ggt tttcaa ggg 864 Ser Trp Asn Thr Val Glu Asp Ser Ile Ile Arg Thr Gly Phe GlnGly 275 280 285 gag agt ggg cac gac ata aaa att act gct gaa aat acc ccgctt cca 912 Glu Ser Gly His Asp Ile Lys Ile Thr Ala Glu Asn Thr Pro LeuPro 290 295 300 atc gcg ggt gtc cta cta ccg act att cct gga aag ctg gacgtt aat 960 Ile Ala Gly Val Leu Leu Pro Thr Ile Pro Gly Lys Leu Asp ValAsn 305 310 315 320 aag tcc aag act cat att tcc gta aat ggt cgg aaa ataagg atg cgt 1008 Lys Ser Lys Thr His Ile Ser Val Asn Gly Arg Lys Ile ArgMet Arg 325 330 335 tgc aga gct ata gac ggt gat gta act ttt tgt cgc cctaaa tct cct 1056 Cys Arg Ala Ile Asp Gly Asp Val Thr Phe Cys Arg Pro LysSer Pro 340 345 350 gtt tat gtt ggt aat ggt gtg cat gcg aat ctt cac gtggca ttt cac 1104 Val Tyr Val Gly Asn Gly Val His Ala Asn Leu His Val AlaPhe His 355 360 365 aga agc agc tcg gag aaa att cat tct aat gaa att tcgtcg gat tcc 1152 Arg Ser Ser Ser Glu Lys Ile His Ser Asn Glu Ile Ser SerAsp Ser 370 375 380 ata ggc gtt ctt ggg tac cag aaa aca gta gat cac accaag gtt aat 1200 Ile Gly Val Leu Gly Tyr Gln Lys Thr Val Asp His Thr LysVal Asn 385 390 395 400 tct aag cta tcg cta ttt ttt gaa atc aaa agc tga1236 Ser Lys Leu Ser Leu Phe Phe Glu Ile Lys Ser 405 410 2 411 PRTArtificial Sequence Description of Artificial Sequence genetic fusion 2Met Gly His His His His His His His His His His Ser Ser Gly His 1 5 1015 Ile Glu Gly Arg Met Ser Gln Ser Asn Arg Glu Leu Val Val Asp Phe 20 2530 Leu Ser Tyr Lys Leu Ser Gln Lys Gly Tyr Ser Trp Ser Gln Phe Ser 35 4045 Asp Val Glu Glu Asn Arg Thr Glu Ala Pro Glu Gly Thr Glu Ser Glu 50 5560 Met Glu Thr Pro Ser Ala Ile Asn Gly Asn Pro Ser Trp His Leu Ala 65 7075 80 Asp Ser Pro Ala Val Asn Gly Ala Thr Ala His Ser Ser Ser Leu Asp 8590 95 Ala Arg Glu Val Ile Pro Met Ala Ala Val Lys Gln Ala Leu Arg Glu100 105 110 Ala Gly Asp Glu Phe Glu Leu Arg Tyr Arg Arg Ala Phe Ser AspLeu 115 120 125 Thr Ser Gln Leu His Ile Thr Pro Gly Thr Ala Tyr Gln SerPhe Glu 130 135 140 Gln Val Val Asn Glu Leu Phe Arg Asp Gly Val Asn TrpGly Arg Ile 145 150 155 160 Val Ala Phe Phe Ser Phe Gly Gly Ala Leu CysVal Glu Ser Val Asp 165 170 175 Lys Glu Met Gln Val Leu Val Ser Arg IleAla Ala Trp Met Ala Thr 180 185 190 Tyr Leu Asn Asp His Leu Glu Pro TrpIle Gln Glu Asn Gly Gly Trp 195 200 205 Asp Thr Phe Val Glu Leu Tyr GlyAsn Asn Ala Ala Ala Glu Ser Arg 210 215 220 Lys Gly Gln Glu Arg Phe AsnArg Trp Phe Leu Thr Gly Met Thr Val 225 230 235 240 Ala Gly Val Val LeuLeu Gly Ser Leu Phe Ser Arg Lys Ala Tyr Ser 245 250 255 Ala Ala Ala HisLys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val 260 265 270 Ser Trp AsnThr Val Glu Asp Ser Ile Ile Arg Thr Gly Phe Gln Gly 275 280 285 Glu SerGly His Asp Ile Lys Ile Thr Ala Glu Asn Thr Pro Leu Pro 290 295 300 IleAla Gly Val Leu Leu Pro Thr Ile Pro Gly Lys Leu Asp Val Asn 305 310 315320 Lys Ser Lys Thr His Ile Ser Val Asn Gly Arg Lys Ile Arg Met Arg 325330 335 Cys Arg Ala Ile Asp Gly Asp Val Thr Phe Cys Arg Pro Lys Ser Pro340 345 350 Val Tyr Val Gly Asn Gly Val His Ala Asn Leu His Val Ala PheHis 355 360 365 Arg Ser Ser Ser Glu Lys Ile His Ser Asn Glu Ile Ser SerAsp Ser 370 375 380 Ile Gly Val Leu Gly Tyr Gln Lys Thr Val Asp His ThrLys Val Asn 385 390 395 400 Ser Lys Leu Ser Leu Phe Phe Glu Ile Lys Ser405 410 3 1704 DNA Artificial Sequence Description of ArtificialSequence genetic fusion 3 atg ggc cat cat cat cat cat cat cat cat catcac agc agc ggc cat 48 Met Gly His His His His His His His His His HisSer Ser Gly His 1 5 10 15 atc gaa ggt cgt cat atg gga acc cca aag cagccc tcg ctg gct cct 96 Ile Glu Gly Arg His Met Gly Thr Pro Lys Gln ProSer Leu Ala Pro 20 25 30 gca cac gcc cta ggc ttg agg aag tcc gat ccc ggaatc cgg agc ctg 144 Ala His Ala Leu Gly Leu Arg Lys Ser Asp Pro Gly IleArg Ser Leu 35 40 45 ggg agc gac gcg gga gga agg cgg tgg aga cca gca gcccag agt atg 192 Gly Ser Asp Ala Gly Gly Arg Arg Trp Arg Pro Ala Ala GlnSer Met 50 55 60 ttc cag atc cca gag ttt gag ccg agt gag cag gaa gac gctagt gct 240 Phe Gln Ile Pro Glu Phe Glu Pro Ser Glu Gln Glu Asp Ala SerAla 65 70 75 80 aca gat agg ggc ctg ggc cct agc ctc act gag gac cag ccaggt ccc 288 Thr Asp Arg Gly Leu Gly Pro Ser Leu Thr Glu Asp Gln Pro GlyPro 85 90 95 tac ctg gcc cca ggt ctc ctg ggg agc aac att cat cag cag ggacgg 336 Tyr Leu Ala Pro Gly Leu Leu Gly Ser Asn Ile His Gln Gln Gly Arg100 105 110 gca gcc acc aac agt cat cat gga ggc gca ggg gct atg gag actcgg 384 Ala Ala Thr Asn Ser His His Gly Gly Ala Gly Ala Met Glu Thr Arg115 120 125 agt cgc cac agt gcg tac cca gcg ggg acc gag gag gat gaa gggatg 432 Ser Arg His Ser Ala Tyr Pro Ala Gly Thr Glu Glu Asp Glu Gly Met130 135 140 gag gag gag ctt agc cct ttt cga gga cgc tcg cgt gcg gct cccccc 480 Glu Glu Glu Leu Ser Pro Phe Arg Gly Arg Ser Arg Ala Ala Pro Pro145 150 155 160 aat ctc tgg gca gcg cag cgc tac ggc cgt gag ctc cga aggatg agc 528 Asn Leu Trp Ala Ala Gln Arg Tyr Gly Arg Glu Leu Arg Arg MetSer 165 170 175 gat gag ttt gag ggt tcc ttc aag gga ctt cct cgc cca aagagc gca 576 Asp Glu Phe Glu Gly Ser Phe Lys Gly Leu Pro Arg Pro Lys SerAla 180 185 190 ggc act gca aca cag atg cga caa agc gcc ggc tgg acg cgcatt atc 624 Gly Thr Ala Thr Gln Met Arg Gln Ser Ala Gly Trp Thr Arg IleIle 195 200 205 cag tcc tgg tgg gat cga aac ttg ggc aaa gga ggc tcc accccc tcc 672 Gln Ser Trp Trp Asp Arg Asn Leu Gly Lys Gly Gly Ser Thr ProSer 210 215 220 cag tca gta ggt agc tca ttg tca tgc ata aat ctt gat tgggat gtc 720 Gln Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp AspVal 225 230 235 240 ata agg gat aaa act aag aca aag ata gag tct ttg aaagag cat ggc 768 Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys GluHis Gly 245 250 255 cct atc aaa aat aaa atg agc gaa agt ccc aat aaa acagta tct gag 816 Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr ValSer Glu 260 265 270 gaa aaa gct aaa caa tac cta gaa gaa ttt cat caa acggca tta gag 864 Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr AlaLeu Glu 275 280 285 cat cct gaa ttg tca gaa ctt aaa acc gtt act ggg accaat cct gta 912 His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr AsnPro Val 290 295 300 ttc gct ggg gct aac tat gcg gcg tgg gca gta aac gttgcg caa gtt 960 Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val AlaGln Val 305 310 315 320 atc gat agc gaa aca gct gat aat ttg gaa aag acaact gct gct ctt 1008 Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr ThrAla Ala Leu 325 330 335 tcg ata ctt cct ggt atc ggt agc gta atg ggc attgca gac ggt gcc 1056 Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile AlaAsp Gly Ala 340 345 350 gtt cac cac aat aca gaa gag ata gtg gca caa tcaata gct tta tcg 1104 Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser IleAla Leu Ser 355 360 365 tct tta atg gtt gct caa gct att cca ttg gta ggagag cta gtt gat 1152 Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly GluLeu Val Asp 370 375 380 att ggt ttc gct gca tat aat ttt gta gag agt attatc aat tta ttt 1200 Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile IleAsn Leu Phe 385 390 395 400 caa gta gtt cat aat tcg tat aat cgt ccc gcgtat tct ccg ggg cat 1248 Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala TyrSer Pro Gly His 405 410 415 aaa acg caa cca ttt ctt cat gac ggg tat gctgtc agt tgg aac act 1296 Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala ValSer Trp Asn Thr 420 425 430 gtt gaa gat tcg ata atc cga act ggt ttt caaggg gag agt ggg cac 1344 Val Glu Asp Ser Ile Ile Arg Thr Gly Phe Gln GlyGlu Ser Gly His 435 440 445 gac ata aaa att act gct gaa aat acc ccg cttcca atc gcg ggt gtc 1392 Asp Ile Lys Ile Thr Ala Glu Asn Thr Pro Leu ProIle Ala Gly Val 450 455 460 cta cta ccg act att cct gga aag ctg gac gttaat aag tcc aag act 1440 Leu Leu Pro Thr Ile Pro Gly Lys Leu Asp Val AsnLys Ser Lys Thr 465 470 475 480 cat att tcc gta aat ggt cgg aaa ata aggatg cgt tgc aga gct ata 1488 His Ile Ser Val Asn Gly Arg Lys Ile Arg MetArg Cys Arg Ala Ile 485 490 495 gac ggt gat gta act ttt tgt cgc cct aaatct cct gtt tat gtt ggt 1536 Asp Gly Asp Val Thr Phe Cys Arg Pro Lys SerPro Val Tyr Val Gly 500 505 510 aat ggt gtg cat gcg aat ctt cac gtg gcattt cac aga agc agc tcg 1584 Asn Gly Val His Ala Asn Leu His Val Ala PheHis Arg Ser Ser Ser 515 520 525 gag aaa att cat tct aat gaa att tcg tcggat tcc ata ggc gtt ctt 1632 Glu Lys Ile His Ser Asn Glu Ile Ser Ser AspSer Ile Gly Val Leu 530 535 540 ggg tac cag aaa aca gta gat cac acc aaggtt aat tct aag cta tcg 1680 Gly Tyr Gln Lys Thr Val Asp His Thr Lys ValAsn Ser Lys Leu Ser 545 550 555 560 cta ttt ttt gaa atc aaa agc tga 1704Leu Phe Phe Glu Ile Lys Ser 565 4 567 PRT Artificial SequenceDescription of Artificial Sequence genetic fusion 4 Met Gly His His HisHis His His His His His His Ser Ser Gly His 1 5 10 15 Ile Glu Gly ArgHis Met Gly Thr Pro Lys Gln Pro Ser Leu Ala Pro 20 25 30 Ala His Ala LeuGly Leu Arg Lys Ser Asp Pro Gly Ile Arg Ser Leu 35 40 45 Gly Ser Asp AlaGly Gly Arg Arg Trp Arg Pro Ala Ala Gln Ser Met 50 55 60 Phe Gln Ile ProGlu Phe Glu Pro Ser Glu Gln Glu Asp Ala Ser Ala 65 70 75 80 Thr Asp ArgGly Leu Gly Pro Ser Leu Thr Glu Asp Gln Pro Gly Pro 85 90 95 Tyr Leu AlaPro Gly Leu Leu Gly Ser Asn Ile His Gln Gln Gly Arg 100 105 110 Ala AlaThr Asn Ser His His Gly Gly Ala Gly Ala Met Glu Thr Arg 115 120 125 SerArg His Ser Ala Tyr Pro Ala Gly Thr Glu Glu Asp Glu Gly Met 130 135 140Glu Glu Glu Leu Ser Pro Phe Arg Gly Arg Ser Arg Ala Ala Pro Pro 145 150155 160 Asn Leu Trp Ala Ala Gln Arg Tyr Gly Arg Glu Leu Arg Arg Met Ser165 170 175 Asp Glu Phe Glu Gly Ser Phe Lys Gly Leu Pro Arg Pro Lys SerAla 180 185 190 Gly Thr Ala Thr Gln Met Arg Gln Ser Ala Gly Trp Thr ArgIle Ile 195 200 205 Gln Ser Trp Trp Asp Arg Asn Leu Gly Lys Gly Gly SerThr Pro Ser 210 215 220 Gln Ser Val Gly Ser Ser Leu Ser Cys Ile Asn LeuAsp Trp Asp Val 225 230 235 240 Ile Arg Asp Lys Thr Lys Thr Lys Ile GluSer Leu Lys Glu His Gly 245 250 255 Pro Ile Lys Asn Lys Met Ser Glu SerPro Asn Lys Thr Val Ser Glu 260 265 270 Glu Lys Ala Lys Gln Tyr Leu GluGlu Phe His Gln Thr Ala Leu Glu 275 280 285 His Pro Glu Leu Ser Glu LeuLys Thr Val Thr Gly Thr Asn Pro Val 290 295 300 Phe Ala Gly Ala Asn TyrAla Ala Trp Ala Val Asn Val Ala Gln Val 305 310 315 320 Ile Asp Ser GluThr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala Leu 325 330 335 Ser Ile LeuPro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly Ala 340 345 350 Val HisHis Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser 355 360 365 SerLeu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp 370 375 380Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu Phe 385 390395 400 Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly His405 410 415 Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp AsnThr 420 425 430 Val Glu Asp Ser Ile Ile Arg Thr Gly Phe Gln Gly Glu SerGly His 435 440 445 Asp Ile Lys Ile Thr Ala Glu Asn Thr Pro Leu Pro IleAla Gly Val 450 455 460 Leu Leu Pro Thr Ile Pro Gly Lys Leu Asp Val AsnLys Ser Lys Thr 465 470 475 480 His Ile Ser Val Asn Gly Arg Lys Ile ArgMet Arg Cys Arg Ala Ile 485 490 495 Asp Gly Asp Val Thr Phe Cys Arg ProLys Ser Pro Val Tyr Val Gly 500 505 510 Asn Gly Val His Ala Asn Leu HisVal Ala Phe His Arg Ser Ser Ser 515 520 525 Glu Lys Ile His Ser Asn GluIle Ser Ser Asp Ser Ile Gly Val Leu 530 535 540 Gly Tyr Gln Lys Thr ValAsp His Thr Lys Val Asn Ser Lys Leu Ser 545 550 555 560 Leu Phe Phe GluIle Lys Ser 565 5 18 DNA Artificial Sequence Description of ArtificialSequence oligonucleotide linker 5 gcgtattctg cggccgcg 18 6 6 PRTArtificial Sequence Description of Artificial Sequence oligopeptidelinker 6 Ala Tyr Ser Ala Ala Ala 1 5 7 1455 DNA Artificial SequenceDescription of Artificial Sequence genetic fusion 7 atg ggc agc agc catcat cat cat cat cac agc agc ggc ctg gtg ccg 48 Met Gly Ser Ser His HisHis His His His Ser Ser Gly Leu Val Pro 1 5 10 15 cgc ggc agc cat atggcg ggc ggt cat ggt gat gta ggt atg cac gta 96 Arg Gly Ser His Met AlaGly Gly His Gly Asp Val Gly Met His Val 20 25 30 aaa gag aaa gag aaa aataaa gat gag aat aag aga aaa gat gaa gaa 144 Lys Glu Lys Glu Lys Asn LysAsp Glu Asn Lys Arg Lys Asp Glu Glu 35 40 45 cga aat aaa aca cag gaa gagcat tta aag gaa atc atg aaa cac att 192 Arg Asn Lys Thr Gln Glu Glu HisLeu Lys Glu Ile Met Lys His Ile 50 55 60 gta aaa ata gaa gta aaa ggg gaggaa gct gtt aaa aaa gag gca gca 240 Val Lys Ile Glu Val Lys Gly Glu GluAla Val Lys Lys Glu Ala Ala 65 70 75 80 gaa aag cta ctt gag aaa gta ccatct gat gtt tta gag atg tat aaa 288 Glu Lys Leu Leu Glu Lys Val Pro SerAsp Val Leu Glu Met Tyr Lys 85 90 95 gca att gga gga aag ata tat att gtggat ggt gat att aca aaa cat 336 Ala Ile Gly Gly Lys Ile Tyr Ile Val AspGly Asp Ile Thr Lys His 100 105 110 ata tct tta gaa gca tta tct gaa gataag aaa aaa ata aaa gac att 384 Ile Ser Leu Glu Ala Leu Ser Glu Asp LysLys Lys Ile Lys Asp Ile 115 120 125 tat ggg aaa gat gct tta tta cat gaacat tat gta tat gca aaa gaa 432 Tyr Gly Lys Asp Ala Leu Leu His Glu HisTyr Val Tyr Ala Lys Glu 130 135 140 gga tat gaa ccc gta ctt gta atc caatct tcg gaa gat tat gta gaa 480 Gly Tyr Glu Pro Val Leu Val Ile Gln SerSer Glu Asp Tyr Val Glu 145 150 155 160 aat act gaa aag gca ctg aac gtttat tat gaa ata ggt aag ata tta 528 Asn Thr Glu Lys Ala Leu Asn Val TyrTyr Glu Ile Gly Lys Ile Leu 165 170 175 tca agg gat att tta agt aaa attaat caa cca tat cag aaa ttt tta 576 Ser Arg Asp Ile Leu Ser Lys Ile AsnGln Pro Tyr Gln Lys Phe Leu 180 185 190 gat gta tta aat acc att aaa aatgca tct gat tca gat gga caa gat 624 Asp Val Leu Asn Thr Ile Lys Asn AlaSer Asp Ser Asp Gly Gln Asp 195 200 205 ctt tta ttt act aat cag ctt aaggaa cat ccc aca gac ttt tct gta 672 Leu Leu Phe Thr Asn Gln Leu Lys GluHis Pro Thr Asp Phe Ser Val 210 215 220 gaa ttc ttg gaa caa aat agc aatgag gta caa gaa gta ttt gcg aaa 720 Glu Phe Leu Glu Gln Asn Ser Asn GluVal Gln Glu Val Phe Ala Lys 225 230 235 240 gct ttt gca tat tat atc gagcca cag cat cgt gat gtt tta cag ctt 768 Ala Phe Ala Tyr Tyr Ile Glu ProGln His Arg Asp Val Leu Gln Leu 245 250 255 tat gca ccg gaa gct ttt aattac atg gat aaa ttt aac gaa caa gaa 816 Tyr Ala Pro Glu Ala Phe Asn TyrMet Asp Lys Phe Asn Glu Gln Glu 260 265 270 ata aat cta tcc atg tct cagagc aac cgg gag ctg gtg gtt gac ttt 864 Ile Asn Leu Ser Met Ser Gln SerAsn Arg Glu Leu Val Val Asp Phe 275 280 285 ctc tcc tac aag ctt tcc cagaaa gga tac agc tgg agt cag ttt agt 912 Leu Ser Tyr Lys Leu Ser Gln LysGly Tyr Ser Trp Ser Gln Phe Ser 290 295 300 gat gtg gaa gag aac agg actgag gcc cca gaa ggg act gaa tcg gag 960 Asp Val Glu Glu Asn Arg Thr GluAla Pro Glu Gly Thr Glu Ser Glu 305 310 315 320 atg gag acc ccc agt gccatc aat ggc aac cca tcc tgg cac ctg gca 1008 Met Glu Thr Pro Ser Ala IleAsn Gly Asn Pro Ser Trp His Leu Ala 325 330 335 gac agc ccc gcg gtg aatgga gcc act gcg cac agc agc agt ttg gat 1056 Asp Ser Pro Ala Val Asn GlyAla Thr Ala His Ser Ser Ser Leu Asp 340 345 350 gcc cgg gag gtg atc cccatg gca gca gta aag caa gcg ctg agg gag 1104 Ala Arg Glu Val Ile Pro MetAla Ala Val Lys Gln Ala Leu Arg Glu 355 360 365 gca ggc gac gag ttt gaactg cgg tac cgg cgg gca ttc agt gac ctg 1152 Ala Gly Asp Glu Phe Glu LeuArg Tyr Arg Arg Ala Phe Ser Asp Leu 370 375 380 aca tcc cag ctc cac atcacc cca ggg aca gca tat cag agc ttt gaa 1200 Thr Ser Gln Leu His Ile ThrPro Gly Thr Ala Tyr Gln Ser Phe Glu 385 390 395 400 cag gta gtg aat gaactc ttc cgg gat ggg gta aac tgg ggt cgc att 1248 Gln Val Val Asn Glu LeuPhe Arg Asp Gly Val Asn Trp Gly Arg Ile 405 410 415 gtg gcc ttt ttc tccttc ggc ggg gca ctg tgc gtg gaa agc gta gac 1296 Val Ala Phe Phe Ser PheGly Gly Ala Leu Cys Val Glu Ser Val Asp 420 425 430 aag gag atg cag gtattg gtg agt cgg atc gca gct tgg atg gcc act 1344 Lys Glu Met Gln Val LeuVal Ser Arg Ile Ala Ala Trp Met Ala Thr 435 440 445 tac ctg aat gac caccta gag cct tgg atc cag gag aac ggc ggc tgg 1392 Tyr Leu Asn Asp His LeuGlu Pro Trp Ile Gln Glu Asn Gly Gly Trp 450 455 460 gat act ttt gtg gaactc tat ggg aac aat gca gca gcc gag agc cga 1440 Asp Thr Phe Val Glu LeuTyr Gly Asn Asn Ala Ala Ala Glu Ser Arg 465 470 475 480 aag ggc cag gaacgc 1455 Lys Gly Gln Glu Arg 485 8 485 PRT Artificial SequenceDescription of Artificial Sequence genetic fusion 8 Met Gly Ser Ser HisHis His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser HisMet Ala Gly Gly His Gly Asp Val Gly Met His Val 20 25 30 Lys Glu Lys GluLys Asn Lys Asp Glu Asn Lys Arg Lys Asp Glu Glu 35 40 45 Arg Asn Lys ThrGln Glu Glu His Leu Lys Glu Ile Met Lys His Ile 50 55 60 Val Lys Ile GluVal Lys Gly Glu Glu Ala Val Lys Lys Glu Ala Ala 65 70 75 80 Glu Lys LeuLeu Glu Lys Val Pro Ser Asp Val Leu Glu Met Tyr Lys 85 90 95 Ala Ile GlyGly Lys Ile Tyr Ile Val Asp Gly Asp Ile Thr Lys His 100 105 110 Ile SerLeu Glu Ala Leu Ser Glu Asp Lys Lys Lys Ile Lys Asp Ile 115 120 125 TyrGly Lys Asp Ala Leu Leu His Glu His Tyr Val Tyr Ala Lys Glu 130 135 140Gly Tyr Glu Pro Val Leu Val Ile Gln Ser Ser Glu Asp Tyr Val Glu 145 150155 160 Asn Thr Glu Lys Ala Leu Asn Val Tyr Tyr Glu Ile Gly Lys Ile Leu165 170 175 Ser Arg Asp Ile Leu Ser Lys Ile Asn Gln Pro Tyr Gln Lys PheLeu 180 185 190 Asp Val Leu Asn Thr Ile Lys Asn Ala Ser Asp Ser Asp GlyGln Asp 195 200 205 Leu Leu Phe Thr Asn Gln Leu Lys Glu His Pro Thr AspPhe Ser Val 210 215 220 Glu Phe Leu Glu Gln Asn Ser Asn Glu Val Gln GluVal Phe Ala Lys 225 230 235 240 Ala Phe Ala Tyr Tyr Ile Glu Pro Gln HisArg Asp Val Leu Gln Leu 245 250 255 Tyr Ala Pro Glu Ala Phe Asn Tyr MetAsp Lys Phe Asn Glu Gln Glu 260 265 270 Ile Asn Leu Ser Met Ser Gln SerAsn Arg Glu Leu Val Val Asp Phe 275 280 285 Leu Ser Tyr Lys Leu Ser GlnLys Gly Tyr Ser Trp Ser Gln Phe Ser 290 295 300 Asp Val Glu Glu Asn ArgThr Glu Ala Pro Glu Gly Thr Glu Ser Glu 305 310 315 320 Met Glu Thr ProSer Ala Ile Asn Gly Asn Pro Ser Trp His Leu Ala 325 330 335 Asp Ser ProAla Val Asn Gly Ala Thr Ala His Ser Ser Ser Leu Asp 340 345 350 Ala ArgGlu Val Ile Pro Met Ala Ala Val Lys Gln Ala Leu Arg Glu 355 360 365 AlaGly Asp Glu Phe Glu Leu Arg Tyr Arg Arg Ala Phe Ser Asp Leu 370 375 380Thr Ser Gln Leu His Ile Thr Pro Gly Thr Ala Tyr Gln Ser Phe Glu 385 390395 400 Gln Val Val Asn Glu Leu Phe Arg Asp Gly Val Asn Trp Gly Arg Ile405 410 415 Val Ala Phe Phe Ser Phe Gly Gly Ala Leu Cys Val Glu Ser ValAsp 420 425 430 Lys Glu Met Gln Val Leu Val Ser Arg Ile Ala Ala Trp MetAla Thr 435 440 445 Tyr Leu Asn Asp His Leu Glu Pro Trp Ile Gln Glu AsnGly Gly Trp 450 455 460 Asp Thr Phe Val Glu Leu Tyr Gly Asn Asn Ala AlaAla Glu Ser Arg 465 470 475 480 Lys Gly Gln Glu Arg 485

We claim:
 1. A functional pro-apoptosis-modifying fusion protein capableof binding a target cell, comprising: (a) a first domain capable ofenhancing apoptosis in the target cell; and (b) a second domain capableof specifically targeting the fusion protein to the target cell, whereinthe fusion protein integrates into or otherwise crosses a cellularmembrane of the target cell upon binding.
 2. The fusion protein of claim1, wherein the first domain is capable of inducing or enhancingapoptosis.
 3. The functional purified apoptosis-modifying fusion proteinof claim 1, comprising an amino acid sequence as set forth as the aminoacid sequence shown in SEQ ID NO: 4 or an amino acid sequence thatdiffers from SEQ ID NO: 4 by one or more conservative amino acidsubstitutions, but which retains targeting and pro-apoptosis-modifyingabilities.
 4. The functional pro-apoptosis-modifying fusion protein ofclaim 1, further comprising: (c) a linker connecting the first domain tothe second domain.
 5. The protein of claim 1, wherein the first domainis a Bcl-2 family protein, or a variant or fragment thereof that retainsan apoptosis-enhancing property.
 6. The protein of claim 5, wherein thefirst domain is Bad, or a variant or fragment thereof that enhancesapoptosis in the target cell to which the protein is exposed.
 7. Theprotein of claim 6, wherein the first domain is a variant of Bad havingan amino acid other than serine at amino acid position 112 and/orposition
 136. 8. The protein of claim 6, wherein the first domainconsists essentially of Bad.
 9. The protein of claim 7, wherein thetarget cell is a tumor cell, a cancer cell, a neoplasm cell, ahyper-proliferative cell, or an adipocyte.
 10. The protein of claim 1,wherein the second domain comprises a receptor-binding domain derivedfrom a bacterial toxin, a monoclonal antibody, a growth factor, or acytokine.
 11. The protein of claim 10, wherein the second binding domaincomprises a receptor-binding domain derived from diphtheria toxin oranthrax toxin.
 12. The protein of claim 10, wherein the second bindingdomain comprises a receptor-binding domain derived from epidermal growthfactor.
 13. The protein of claim 10, wherein the receptor-binding domaincomprises diphtheria toxin receptor binding domain, or a variant orfragment thereof that targets the fusion protein to the target cell towhich the protein is exposed.
 14. The protein of claim 10, wherein thesecond domain further comprises a translocation domain of diphtheriatoxin.
 15. The protein of claim 4, wherein the linker is 5-100 aminoacid residues in length.
 16. The protein of claim 4, wherein the linkercomprises the amino acid sequence shown in SEQ ID NO:
 6. 17. The proteinof claim 16, wherein the linker consists of the amino acid sequenceshown in SEQ ID NO:
 6. 18. The functional apoptosis-modifying fusionprotein of claim 1, comprising: (a) Bad; (b) a diphtheria toxintranslocation domain; and (c) a bacterial toxin receptor binding domain,wherein (a), (b), and (c) are functionally linked.
 19. The fusionprotein of claim 18, wherein (c) is a diphtheria toxin or anthrax toxinreceptor binding domain.
 20. A composition comprising the proteinaccording to claim
 1. 21. A pharmaceutical composition comprising thecomposition according to claim 20, and a pharmaceutically acceptablecarrier.
 22. A combined pharmaceutical composition comprising a fusionprotein according to claim 19, and a sufficient amount of PA to enablemeasurable transport of the fusion protein into a target cell.
 23. Theprotein of claim 1 for use in enhancing apoptosis in a target cell.